Method of administration and treatment

ABSTRACT

Provided herein are formulations for topical and/or transdermal administration, and methods of using these formulations for the treatment of proliferative diseases related to cancer such as cancers and related conditions, and solid tumors. Also provided are formulations for topical and/or transdermal administration, and methods of using these formulations for melasma, gout, skin disorders, and other diseases and disorders described herein as well as methods for modulating the pH (e.g. raising) of a tissue or microenvironment proximal to a tumor, modulating pH or improving the effectiveness of know chemotherapeutic agents, immunotherapy and the like for the prevention, treatment of cancers and related conditions described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/866,466 filed May 4, 2020, which claims priority to U.S. patentapplication Ser. No. 16/546,256 filed Aug. 20, 2019, which claimspriority to U.S. patent application Ser. No. 16/132,258 filed Sep. 14,2018 and U.S. patent application Ser. No. 16/132,357 filed Sep. 14,2018, which are related to and claims priority to International patentApplication PCT/US18/28017 by Bruce Sand filed Apr. 17, 2018, entitled‘Parental non-systemic administration of buffering agents for inhibitingmetastasis of solid tumors, hyperpigmentation and gout’, U.S.Provisional Application Ser. No. 62/559,947 filed Sep. 18, 2017 entitled‘Parental non-systemic administration of buffering agents for treatmentof cancer’, provisional applications U.S. Ser. No. 62/559,947 filed 18Sep. 2017; U.S. Ser. No. 62/559,360 filed 15 Sep. 2017; U.S. Ser. No.62/562,725 filed 25 Sep. 2017; U.S. Ser. No. 62/609,982 filed 22 Dec.2017; and U.S. Ser. No. 62/639,904 filed 7 March, all incorporated byreference in their entirety herein.

FIELD OF INVENTION

This invention relates generally to methods of treatment and therapeuticuses for enhanced formulations for transdermal or topical delivery oftherapeutic agents.

BACKGROUND

The following includes information that may be useful in understandingthe present inventions. It is not an admission that any of theinformation provided herein is prior art, or relevant, to the presentlydescribed or claimed inventions, or that any publication or documentthat is specifically or implicitly referenced is prior art.

Progression to metastasis remains the highest mortality risk for cancerpatients, despite significant efforts to therapeutically targetmetastatic lesions. Tumor invasion and metastasis associated withneoplastic progression are the major causes of cancer deaths andunderstanding the mechanisms determining metastatic spread of malignantcells via invasion to distant tissues is, perhaps, the central questionin oncology.

It is known that microenvironmental acidosis in a primary tumorincreases cellular motility and invasiveness, leading to increasedmetastasis and that solid tumors exist in a microenvironment ofrelatively low pH, presumably because of the hypoxic nature of suchtumors, increased glycolytic metabolism of glucose and poor perfusion.As early as 1979, Turner, G A, Experientia (1979) 35:1657-1658 reportedthat acid pH encouraged the release of tumor cells by collagenase, thusencouraging metastases. Curvier, C. et al., Clin. Exp Metastasis (1997)15:19-25 reported enhanced invasive capacity of tumor cells due toglucose starvation, hypoxia and acidosis. Rofstad, E. K. et al., CancerRes (2006) 66:6699-6706 reported that the acidic extracellular pH ofhuman melanoma cells promoted metastasis in mice.

The extracellular pH of malignant solid tumors is acidic, in the rangeof 6.5 to 6.9, whereas the pH of normal tissues is significantly morealkaline, 7.2 to 7.5. These observations have led to the “acid-mediatedinvasion hypothesis,” wherein tumor-derived acid facilitates tumorinvasion by promoting normal cell death and extracellular matrix (ECM)degradation of the parenchyma surrounding growing tumors.

According to miscellaneous sources in the popular press, certainphysicians have experimented with intravenous sodium bicarbonate as amethod of inhibiting metastatic cancer. A series of articles by Robey,I. F. et al., beginning with a publication in Cancer Res (2009)69:2260-2267 demonstrated that oral bicarbonate reduces the formation ofspontaneous metastases in mouse models of metastatic breast cancer.Additional publications such as Robey, I. F. et al., BMC Cancer (2011)11:235-245 and Robey, I. F., et al. BioMed Res International (2013)pages 1-10 and Robey, I. F. et al., J. Integr. Uncol. (2015) 4:1-8described oral administration of bicarbonate as an inhibitor ofmetastases first in mice, and then in human volunteers. Ribeiro, M. deL., et al., J. Nutr Food Sci (2013) 2:6-16 assigned various pH scores tofoods (including wine) and also described oral administration of lysinebuffer and bicarbonate in mice bearing prostate cancer. In addition,Silva, A. S. et al., Cancer Res (2009) 2677-2684 described the role ofsystemic buffers in reducing metastases. Sircus, M. published a book,Sodium Bicarbonate: Nature's Unique First Aid Remedy, Garden City Park,N.Y.: Square One Publishers, 2014, advocating sodium bicarbonate as aremedy for various conditions.

Thus, the idea of using buffers to modulate the pH of the tissuessurrounding tumors is not a new concept, however in practice it hasfailed. A primary reason for this failure is that oral and conventionaladministration of buffers has serious limitations inpractice—specifically intolerance and side effects including diarrhea,gastric intolerance, nausea, vomiting and abdominal discomfort.Therapeutically effective amounts of pharmaceutical formulationscomprising pH modulating buffers and the like cannot be administered,delivered, and tolerated orally. Intravenous i.e. systemicadministration has also been discredited. Also, it is not clear whatcancers would respond to an adjustment of microenvironmental pH and whatbuffers would be effective. The possibility that other metabolicprocesses, pathways and mechanisms of action, and the like that areancillary or independent of adjusting the microenvironmental pH may beinvolved warrants further study and development.

New treatments, formulations and methods of administration of buffers(e.g. sodium bicarbonate and others) need to be developed that overcomethe current deficiencies in such buffering formulations in order todetermine the efficacy of various buffering formulations on thetreatment of cancer, gout, immunological disorders, skin disorders, andother diseases and disorders described herein. For example, also neededare buffering formulations for topical administration and methods toinhibit cancer or prevent the metastasis, intravasion, invasion, and thegrowth of cancer cells or tumors. Also lacking in the art are effectivebuffering formulations and methods for topical administration for theprevention of cancers, for maintaining remission, and for palliativecare. Another area that the inventors believe is unmet is formulationsof one or more buffering agent and methods of use in combination withother agents or treatments such as chemotherapeutics,immunotherapeutics, or other bioactive agents or Biologics such asantibody-based therapies or therapeutics. The inventions describedherein address these unmet needs.

In another aspect of the invention, new treatments, formulations andmethods of administration of buffers (e.g. sodium bicarbonate andothers) need to be developed for the treatment of gout/hyperuricaemia.Gout is a major health problem worldwide, with the prevalence varyingfrom 0.1% to 10% in different regions. A National Health and NutritionExamination Survey 2007-2008 showed that among adults aged over 20 yearsin the United States, 3.9% had self-reported gout, while only 2.9% ofthe population reported gout in the 1988-1994 survey. In mainland China,a systematic review of data from 2000 to 2014 suggested the prevalenceof hyperuricaemia and gout in the general population were 13.3% and1.1%, respectively. In general, both developed and developing countriespresented with increasing prevalence and incidence of gout in recentdecades. Patients with hyperuricaemia or gout are at risk of developinga variety of comorbidities, such as hypertension, chronic kidneydisease, cardiovascular diseases, metabolic syndromes and psychiatricdisorders. A recent survey found that 5%-10% of patients with gout hadat least seven comorbidities and that hypertension was presented in atleast 74% patients with gout. Li nQ., Li X., Kwong J. S-W. et al., BMJOpen, 2017, 7:e014928.doi:10.1136/bmjopen-2016-014928. Unfortunately,gout remains under-diagnosed and under-treated in the general community.Despite major advances in treatment strategies, as many as 90% ofpatients with gout are poorly controlled or improperly managed and theirhyperuricemia and recurrent flares continue. Igel., T. F., et al.,Recent advances in understanding and managing gout, F1000Res., 2017 Mar.10; 6:247. doi: 10.12688/f1000research.9402.1. e Collection 2017. Thus,it appears that current treatment approaches have failed to make animpact on the treatment of gout. Further inventions described hereinaddress this unmet need.

SUMMARY

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Brief Summary. The inventions described andclaimed herein are not limited to, or by, the features or embodimentsidentified in this Summary, which is included for purposes ofillustration only and not restriction.

Applicants have found that the drawbacks of intravenous and oraladministration of buffers and other anti-metastatic agents can beovercome by administering these agents topically and/or transdermally,but other types of administration are possible, including for example,intranasally or via transmembrane administration for example bysuppository or intranasal application.

Accordingly, in one aspect a method of treating a proliferative disorderassociated with cancer in a patient is provided. In some embodiments themethod comprises administering topically and/or transdermally aneffective amount of a formulation for transdermal delivery comprisingone or more buffering agent to a patient in need thereof, where theadministration is effective to i) inhibit or prevent the growth of atumor or tumor cells, ii) inhibit or prevent the metastasis of tumors orcancer cells, iii) inhibit or prevents carcinogenesis, iv) inhibit orprevent the intravasation of tumor cells, or v) improve or extend theduration of remission, or maintain remission of a cancer or tumor.

A proliferative disorder associated with cancer may include anycondition, disease, disorder, cellular or metabolic state that isassociated with carcinogenesis or a cancer, tumor, cancer cells, or thelike. In certain embodiments, treating a proliferative disorder inhibitsor prevents the growth of a tumor or tumor cells. In certainembodiments, treating a proliferative disorder inhibits or prevents themetastasis of tumors or cancer cells. In certain embodiments, treating aproliferative disorder inhibits or prevents carcinogenesis. In certainembodiments, treating a proliferative disorder inhibits or prevents theintravasation of tumor cells. In certain embodiments, treating aproliferative disorder improves or extends the duration of remission ormaintains remission of a cancer or tumor.

In one aspect, a method of treating cancer in a patient is providedcomprising administering topically and/or transdermally an effectiveamount of a formulation comprising one or more buffering agent to apatient in need thereof, where the administration is effective toinhibit or prevent the growth of a tumor or tumor cells.

In one aspect, a method of preventing or inhibiting the metastasis oftumors is provided comprising administering topically and/ortransdermally an effective amount of a formulation comprising one ormore buffering agent to a patient in need thereof, where theadministration is effective to inhibit or prevents the metastasis oftumors or cancer cells.

In one aspect, a method of preventing the intravasation of tumor cellsis provided comprising administering topically and/or transdermally aneffective amount of a formulation comprising one or more buffering agentto a patient in need thereof, where the administration is effective toinhibit or prevent the intravasation of tumor cells.

In another aspect, a method of improving, extending the duration ofremission, or maintaining remission of a cancer or tumor is providedcomprising administering topically and/or transdermally an effectiveamount of a formulation comprising one or more buffering agent to apatient in need thereof, where the administration is effective inimproving, extending the duration of remission, or maintaining remissionof a cancer or tumor.

In another aspect, a method of preventing carcinogenesis is providedcomprising administering topically and/or transdermally an effectiveamount of a formulation comprising one or more buffering agent to apatient in need thereof, where the administration is effective toinhibit or prevent carcinogenesis. In another aspect, embodiments areprovided herein for preventing or inhibiting carcinogenesis that includethe administration of another anti-cancer agent. An exemplary embodimentof this aspect is a method of preventing or inhibiting carcinogenesiscomprising i) selecting a therapeutic agent (e.g. a biological agent,chemotherapeutic or immunotherapeutic agent), ii) formulating thetherapeutic agent in a suitable formulation, iii) administering theformulation comprising the therapeutic agent, and iv) before, during orafter step iii), administering a formulation comprising one or morebuffering agent topically and/or transdermally in an amount effective toinhibit or prevent carcinogenesis.

In another aspect, a method of maintaining remission of a cancer isprovided comprising administering topically and/or transdermally aneffective amount of a formulation comprising one or more buffering agentto a patient in need thereof, where the administration is effective tomaintain remission. In another aspect, embodiments are provided hereinfor maintaining remission that include the administration of otheragents (e.g. an anti-cancer agent). An exemplary embodiment of thisaspect is a method of maintaining remission of a cancer comprising i)selecting a therapeutic agent (e.g. a biological agent, chemotherapeuticor immunotherapeutic agent), ii) formulating the therapeutic agent in asuitable formulation, iii) administering the formulation comprising thetherapeutic agent, and iv) before, during or after step iii),administering a formulation comprising one or more buffering agenttopically and/or transdermally in an amount effective to maintainremission of a cancer.

In another aspect, a method of treating cancer in a patient is providedwhere an effective amount of a formulation comprising one or morebuffering agent is administering topically and/or transdermally to apatient in need thereof such that the administration is effective toalter the pH of a tissue or microenvironment proximal to a solid tumoror cancer cells in the patient. In certain embodiments, the change inthe pH of a tissue or microenvironment proximal to a solid tumor orcancer cells inhibits the growth of said solid tumor or cancer cells.

In another aspect, a method of altering the pH of a tissue ormicroenvironment proximal to a solid tumor or cancer cells in a patientis provided where an effective amount of a formulation comprising one ormore buffering agent is administering topically and/or transdermally toa patient in need thereof such that the administration is effective toalter the pH of a tissue or microenvironment proximal to a solid tumoror cancer cells in the patient.

In another aspect, a method of preventing metastasis of tumors isprovided where an effective amount of a formulation comprising one ormore buffering agent is administering topically and/or transdermally toa patient in need thereof such that the administration is effective toalter the pH of a tissue or microenvironment proximal to a solid tumoror cancer cells in the patient and the change in the pH of a tissue ormicroenvironment proximal to a solid tumor or cancer cells inhibits orprevents the metastasis of tumors or cancer cells.

In another aspect, a method of preventing the intravasation of tumorcells is provided where an effective amount of a formulation comprisingone or more buffering agent is administering topically and/ortransdermally to a patient in need thereof such that the administrationis effective to alter the pH of a tissue or microenvironment proximal toa solid tumor or cancer cells in the patient and the change in the pH ofa tissue or microenvironment proximal to a solid tumor or cancer cellsinhibits or prevents the intravasation of tumor cells.

In another aspect, a method of treatment of cancer is providedcomprising i) selecting a therapeutic agent (e.g. a chemotherapeutic orimmunotherapeutic agent), ii) formulating the therapeutic agent in asuitable formulation, iii) administering the formulation comprising thetherapeutic agent, and iv) before, during or after step iii),administering a formulation comprising one or more buffering agenttopically and/or transdermally in an amount effective to inhibit orprevent the growth of a tumor or tumor cells. The therapeutic agent istypically formulated in a formulation suitable for a route ofadministration other than topical and/or transdermal, however in certainembodiments the therapeutic agent is formulated with the buffering agentin the same formulation.

In another aspect, methods of increasing the efficacy of conventionalapproaches to cancer treatment are provided. In some embodiments,methods of increasing the efficacy of immunotherapy are providedcomprising administering i) a formulation comprising one or morebuffering agent topically and/or transdermally, and ii) before, duringor after step i), administering a formulation comprising aimmunotherapeutic agent. In some embodiments, methods of increasing theefficacy of biological therapeutic agents are provided comprisingadministering i) a formulation comprising one or more buffering agenttopically and/or transdermally, and ii) before, during or after step i),administering a formulation comprising a selected biological therapeuticagent. In other embodiments, methods of increasing the efficacy ofchemotherapy are provided comprising administering i) a formulationcomprising one or more buffering agent topically and/or transdermally,and ii) before, during or after step i), administering a formulationcomprising a chemotherapeutic agent.

In another aspect, methods of palliative care for terminally ill cancersubjects are provided comprising administering topically and/ortransdermally an effective amount of a formulation comprising one ormore buffering agent to a patient in need thereof, where theadministration is effective to improve the quality of life of aterminally ill patient.

In another aspect, a method of treating gout is provided where aneffective amount of a formulation comprising one or more buffering agentis administering topically and/or transdermally to a patient in needthereof such that the administration is effective to treat or reduce thesymptoms of gout in said patient.

In another aspect, a method of treating a skin disorder in a subject isprovided where an effective amount of a formulation comprising one ormore buffering agent is administering topically and/or transdermally toa patient in need thereof such that the administration is effective totreat or reduce the skin disorder symptoms in said patient.

In another aspect, a method of treating melasma in a subject is providedwhere an effective amount of a formulation comprising one or morebuffering agent is administering topically and/or transdermally to apatient in need thereof such that the administration is effective totreat or reduce the melasma symptoms in said patient.

In another aspect, a method of improving, extending the duration ofremission, or maintaining remission of a cancer or tumor is providedwhere an effective amount of a formulation comprising one or morebuffering agent is administering topically and/or transdermally to apatient in need thereof. In certain embodiments the administration iseffective to alter the pH of a tissue or microenvironment proximal to asolid tumor or cancer cells in the patient and the change in the pH of atissue or microenvironment proximal to a solid tumor or cancer cells toimprove, extend the duration of remission, or maintain remission of acancer or tumor.

In another aspect, the invention is directed to a method to inhibitmetastasis and/or growth of a solid tumor contained in a subject whichmethod comprises administering by a topical and/or transdermal route ofadministration to a subject in need of such inhibition an effectiveamount of an anti-metastasis agent. In some embodiments, theanti-metastasis agent may be buffer sufficient to increase the pH of themicroenvironment of the solid tumor or cancer cells, or a proteaseinhibitor, an inhibitor of Na⁺/H⁺ exchanger activity, an inhibitor ofepidermal growth factor receptor (EGFR), an inhibitor of src homologyregion 2-containing protein tyrosinase phosphatase (Shp2), with aferin Aor combinations thereof. The administration is by non-systemicparenteral route includes topical administration, especiallytransdermal.

For transdermal topical administration in particular for agents otherthan buffer, a suitable formulation typically involves a penetrant thatenhances penetration of the skin and is, in some embodiments, composedof chemical permeation enhancers (CPEs). In some cases, it can alsoinclude peptides designed to penetrate cells i.e. cell penetratingpeptides (CPPs) also known as skin penetrating peptides (SPPs). Theformulation may be applied for example in the form of topical lotions,creams, and the like, as described herein.

If the active agent is a buffer, the choice of buffer system is based onthe criteria of capability of buffering at a suitable pH typicallybetween 7 and 10.5, as well as biocompatibility of the buffer systemitself and the compatibility of the buffer system with the remainingcomponents of the formulation. Conversely, the formulation is chosen tobe compatible with the buffer selected; amounts of penetrants aregenerally less than those advantageous for therapeutic agents ingeneral.

Some tumors may be resistant to lower doses, but treatable with higherdoses. When the pH is adjusted for the purpose of inhibiting metastasis,treatment is followed by assessment of effectiveness. More importantly,as noted above, it has been found that some tumors are resistant tobuffer treatment—i.e., raising the pH does not have a metastasisinhibiting effect. See, for example, Bailey, K M, et al., Neoplasia(2014) 16:354-364 (supra). It is therefore one aspect of the inventionto evaluate the tumors of potential subjects for treatment with buffer,e.g., by culturing biopsies for sensitivity/resistance to pH adjustment.In a related aspect, particular types of tumors are evaluated forsensitivity/resistance to pH adjustment and capacity for treatment as afunction of dosage and buffer formulation composition,

As described in more detail herein, raising the pH in the vicinity ofother diseases, disorders, and conditions can also an effectivetreatment. For example, raising the pH in the vicinity of melasmas andgout is also an effective treatment. The methods of the invention canusefully be applied to treatment of these conditions as well. Thus,another aspect of the invention such methods of treatment directed tomelasma and gout.

Other features and advantages of aspects of the present invention willbecome apparent from the following more detailed description, taken inconjunction with the accompanying drawings, which illustrate, by way ofexample, the principles of aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate aspects of the present invention.In such drawings:

FIG. 1 shows the time course of urine pH immediately following topicaladministration of sodium bicarbonate in various formulations and dosageregimes.

FIG. 2 shows the mean daily urine pH of experimental subjects as afunction of time after administration of a formulation of sodiumbicarbonate in the formulations and dosages of FIG. 1.

FIG. 3 shows the time course of urine pH over a 3-day period usingalternative topical formulations.

FIG. 4 shows the % change in target joint pain scores measured startingat 15 min and over a 14 day period.

FIG. 5 shows the target joint pain scores on a scale of 0-8, measuredstarting at 15 min and over a 14 day period.

FIG. 6 shows the time course of urine pH over a 3-day period using andcomparing four different topical formulations.

FIG. 7 shows the time course of urine pH over a 3-day period using andcomparing three different topical formulations.

FIG. 8 shows the time course of urine pH over a 3-day period using andcomparing four different topical formulations.

FIG. 9 shows the time course of urine pH over a 3-day period using oneformulation applied topically at three doses.

FIG. 10 shows the time course of urine pH over a 3-day period using oneformulation applied topically at three doses.

FIG. 11 is a dose response curve showing urine pH for 3 days, where the200 mM sodium bicarbonate control is shown in yellow, the 100 μl offormulation is grey, the 125 μl of formulation is orange, and the doseamount of 150μl of formulation is blue.

FIG. 12 shows urine pH measured over 3 days for two pH modulatingbuffering formulations: A, B and 200 mM sodium bicarbonate in wateradministered as libitum as a control.

FIG. 13 shows urine pH measured over 3 days for three pH modulatingbuffering formulations: A, #25, #28, #29 and 200 mM sodium bicarbonatein water administered as libitum as a control.

FIG. 14 shows urine pH measured over 3 days for three pH modulatingbuffering formulations: #26 and #27.

FIG. 15 shows urine pH measured over 3 days for four pH modulatingbuffering formulations: #22A, #22B, #23, and #24.

FIG. 16—shows the regulation of NHE1 and its roles in driving tumorbehaviors.

FIG. 17—illustrates the development of tumor metabolic microenvironment.

FIG. 18—depicts a table of gout joint pain measurements.

FIG. 19—depicts a table of gout joint pain measurements with the %change from baseline for each timepoint

DETAILED DESCRIPTION

The practices described herein employ, unless otherwise indicated,conventional techniques of tissue culture, immunology, molecularbiology, microbiology, cell biology and recombinant DNA, which arewithin the skill of the art. See, e.g., Harlow and Lane eds. (1999)Antibodies, A Laboratory Manual and Herzenberg et al. eds (1996) Weir'sHandbook of Experimental Immunology.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are to be understood asapproximations in accordance with common practice in the art. When usedherein, the term “about” may connote variation (+) or (−) 1%, 5% or 10%of the stated amount, as appropriate given the context. It is to beunderstood, although not always explicitly stated, that the reagentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a pharmaceutically acceptable carrier”includes a plurality of pharmaceutically acceptable carriers, includingmixtures thereof. On the other hand “one” designates the singular.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the listed elements, but do not excludeother unlisted elements. “Consisting essentially of” when used to definecompositions and methods, excludes other elements that alters the basicnature of the composition and/or method, but does not exclude otherunlisted elements. Thus, a composition consisting essentially of theelements as defined herein would not exclude trace amounts of elements,such as contaminants from any isolation and purification methods orpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like, but would exclude additional unspecifiedamino acids. “Consisting of” excludes more than trace elements of otheringredients and substantial method steps for administering thecompositions described herein. Embodiments defined by each of thesetransition terms are within the scope of this disclosure and theinventions embodied therein.

As noted above, one aspect of the invention is a method to inhibitcancer growth and metastasis, including diminution of cancer mass bynon-systemic parenteral, including topical administration ofantimetastatic agents, including those agents that result in bufferingthe immediate environment of tumor cells, including solid tumors andmelanomas. For non-systemic parenteral administration, such asintramuscular, intraperitoneal or subcutaneous administration standardformulations are sufficient. These formulations include standardexcipients and other ancillary ingredients such as antioxidants,suitable salt concentrations and the like. Such formulations can befound, for example, in Remington's Pharmaceutical Sciences (13^(th) Ed),Mack Publishing Company, Easton, Pa.—a standard reference for varioustypes of administration. As used herein, the term “formulation(s)” meansa combination of at least one active ingredient with one or more otheringredient, also commonly referred to as excipients, which may beindependently active or inactive. The term “formulation”, may or may notrefer to a pharmaceutically acceptable composition for administration tohumans or animals, and may include compositions that are usefulintermediates for storage or research purposes. In an embodiment,administration to humans or animals may include, without limitation,topical, sublingual, rectal, vaginal, transdermal, trancutaneous, oral,inhaled, intranasal, pulmonary, subcutaneous, pulmonary, intravenous,enteral or parenteral. Suitable topical formulations for transdermaladministration of active agents for the methods provided herein aredescribed in U.S. Ser. No. 14/757,703, to Sand B., et al., incorporatedherein by reference in it's entirety. Suitable penetrants are described,for example, in PCT publications WO/2016/105499 and WO/2017/127834.

As the patients and subjects of the invention method are, in addition tohumans, veterinary subjects, formulations suitable for these subjectsare also appropriate. Such subjects include livestock and pets as wellas sports animals such as horses, greyhounds, and the like.

In an embodiment, a “pharmaceutical composition” is intended to include,without limitation, the combination of an active agent with a carrier,inert or active, in a sterile composition suitable for diagnostic ortherapeutic use in vitro, in vivo or ex vivo. In one aspect, thepharmaceutical composition is substantially free of endotoxins or isnon-toxic to recipients at the dosage or concentration employed.

In an embodiment, “an effective amount” refers, without limitation, tothe amount of the defined component sufficient to achieve the desiredchemical composition or the desired biological and/or therapeuticresult. In an embodiment, that result can be the desired pH or chemicalor biological characteristic, e.g., stability of the formulation. Inother embodiments, the desired result is the alleviation or ameliorationof the signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. When the desired result is atherapeutic response, the effective amount will, without limitation,vary depending upon the specific disease or symptom to be treated oralleviated, the age, gender and weight of the subject to be treated, thedosing regimen of the formulation, the severity of the diseasecondition, the manner of administration and the like, all of which canbe determined readily by one of skill in the art. A desired effectedmay, without necessarily being therapeutic, also be a cosmetic effect,in particular for treatment for disorders of the skin described herein.

In an embodiment, a “subject” of diagnosis or treatment is, withoutlimitation, a prokaryotic or a eukaryotic cell, a tissue culture, atissue or an animal, e.g. a mammal, including a human. Non-human animalssubject to diagnosis or treatment include, for example, withoutlimitation, a simian, a murine, a canine, a leporid, such as a rabbit,livestock, sport animals, and pets.

In an embodiment, as used herein, the terms “treating,” “treatment” andthe like are used herein, without limitation, to mean obtaining adesired pharmacologic and/or physiologic effect. The effect may beprophylactic in terms of completely or partially preventing a disorderor sign or symptom thereof, and/or may be therapeutic in terms ofamelioration of the symptoms of the disease or infection, or a partialor complete cure for a disorder and/or adverse effect attributable tothe disorder.

Methods

Methods for treating, preventing or ameliorating a disease, disorder, acondition, or a symptom thereof or a condition related thereto areprovided herein using formulations for transdermal delivery describedherein below. The methods provided herein may comprise or consist oftopically administering one or more of the formulations described hereinto skin of a subject in need thereof. Preferred, but non-limitingembodiments are directed to methods for treating, preventing, inhibitingor ameliorating a disease, disorder, a condition, or a symptom describedbelow.

Caners and Tumors

Many embodiments provided herein are directed to various methods oftreating cancer and/or tumors. An exemplary embodiment of a method oftreating cancer in a patient according to the invention comprisesadministering topically and/or transdermally an effective amount of aformulation comprising one or more buffering agent to a patient in needthereof, wherein said administration is effective to inhibit or preventthe growth of a tumor or tumor cells.

Another embodiment is directed to a method of preventing metastasis oftumors comprising administering topically and/or transdermally aneffective amount of a formulation comprising one or more buffering agentto a patient in need thereof, where the administration is effective toinhibit or prevents the metastasis of tumors or cancer cells.

Another embodiment is directed to a method of preventing theintravasation of tumor cells comprising administering topically and/ortransdermally an effective amount of a formulation comprising one ormore buffering agent to a patient in need thereof, where theadministration is effective to inhibit or prevent the intravasation oftumor cells.

Another embodiment is directed to a method of treatment of cancer, themethod comprising i) selecting a therapeutic agent (e.g. achemotherapeutic of immunotherapeutic agent) described herein andformulating the therapeutic agent in a formulation comprising one ormore buffering agent, and iii) administering the formulation topicallyand/or transdermally in an amount effective to inhibit or prevent thegrowth of a tumor or tumor cells.

Another embodiment is directed to a method of improving, extending theduration of remission, or maintaining remission of a cancer or tumorcomprising administering topically and/or transdermally an effectiveamount of a formulation comprising one or more buffering agent to apatient in need thereof, where administration is effective to improve,extend the duration of remission, or maintain remission of a cancer ortumor.

In other embodiments, a method of treating cancer in a patient comprisesadministering topically and/or transdermally an effective amount of aformulation comprising one or more buffering agent to a patient in needthereof, where the administration is effective to alter the pH of atissue or microenvironment proximal to a solid tumor or cancer cells inthe patient, wherein the change in the pH of a tissue ormicroenvironment proximal to a solid tumor or cancer cells inhibits thegrowth of said solid tumor or cancer cells.

In other embodiments, a method of altering the pH of a tissue ormicroenvironment proximal to a solid tumor or cancer cells in a patientis provided. These embodiments generally comprise administeringtopically and/or transdermally an effective amount of a formulationcomprising one or more buffering agent to a patient in need thereof,wherein the administration is effective to alter the pH of a tissue ormicroenvironment proximal to a solid tumor or cancer cells in thepatient.

In other embodiments, a method of inhibiting or preventing themetastasis of tumors in a patient is provided. These embodimentsgenerally comprise administering topically and/or transdermally aneffective amount of a formulation comprising one or more buffering agentto a patient in need thereof, wherein the administration is effective toalter the pH of a tissue or microenvironment proximal to a solid tumoror cancer cells in the patient, and where the change in the pH of atissue or microenvironment proximal to a solid tumor or cancer cellsinhibits or prevents the metastasis of tumors or cancer cells.

In other embodiments, a method of inhibiting or preventing theintravasation of tumor cells in a patient is provided. These embodimentsgenerally comprise administering topically and/or transdermally aneffective amount of a formulation comprising one or more buffering agentto a patient in need thereof, wherein the administration is effective toinhibit or prevent the intravasation of tumor cells.

Formulations provided herein are used in methods of treating manycancers, including but not limited to breast cancer, prostate cancer,pancreatic cancer, lung cancer, bladder cancer, skin cancer, colorectalcancer, kidney cancer, liver cancer, and thyroid cancer.

Formulations provided herein are also used in methods of treating acancer or tumor, including but not limited to Adrenocortical Carcinoma,Basal Cell Carcinoma, Bladder Cancer, Bone Cancer, Brain Tumor, BreastCancer, Cervical Cancer, Colon Cancer, Colorectal Cancer, EsophagealCancer, Retinoblastoma, Gastric (Stomach) Cancer, GastrointestinalTumors, Glioma, Head and Neck Cancer, Hepatocellular (Liver) Cancer,Islet Cell Tumors (Endocrine Pancreas), Kidney (Renal Cell) Cancer,Laryngeal Cancer, Non-small Cell Lung Cancer, Small Cell Lung Cancer,Medulloblastoma, Melanoma, Pancreatic Cancer, Prostate Cancer, RenalCancer, Rectal cancer, and Thyroid Cancer.

While preferred embodiments of the methods provided herein are typicallydirected to a particular cancer, solid tumor or grouping thereof, a morecomplete but still non-limiting listing of suitable cancers and tumorsthat may be tested for effectiveness according to embodiments providedherein includes the following: lymphoblastic leukemia (ALL), acutemyeloid leukemia (AML), adrenocortical carcinoma, aids-related cancers,kaposi sarcoma (soft tissue sarcoma), aids-related lymphoma (lymphoma),primary cns lymphoma (lymphoma), anal cancer, astrocytomas, atypicalteratoid/rhabdoid tumor, childhood, central nervous system (braincancer), basal cell carcinoma, bile duct cancer, bladder cancer.childhood bladder cancer, bone cancer (includes ewing sarcoma andosteosarcoma and malignant fibrous histiocytoma), brain tumors, breastcancer, childhood breast cancer, bronchial tumors, burkitt lymphoma(non-hodgkin lymphoma, carcinoid tumor (gastrointestinal), childhoodcarcinoid tumors, cardiac (heart) tumors, central nervous system tumors.atypical teratoid/rhabdoid tumor, childhood (brain cancer), embryonaltumors, childhood (brain cancer), germ cell tumor (childhood braincancer), primary cns lymphoma, cervical cancer, childhood cervicalcancer, cholangiocarcinoma, chordoma (childhood), chronic lymphocyticleukemia (CLL), chronic myelogenous leukemia (cml), chronicmyeloproliferative neoplasms, colorectal cancer, childhood colorectalcancer, craniopharyngioma (childhood brain cancer), cutaneous t-celllymphoma, ductal carcinoma in situ (DCIS), embryonal tumors, (childhoodbrain CNS cancers), endometrial cancer (uterine cancer), ependymoma,esophageal cancer, childhood esophageal cancer, esthesioneuroblastoma(head and neck cancer), Ewing sarcoma (bone cancer), extracranial germcell tumors, extragonadal germ cell tumors, eye cancer, childhoodintraocular melanoma, intraocular melanoma, retinoblastoma, fallopiantube cancer, fibrous histiocytoma of bone (malignant, and osteosarcoma),gallbladder cancer, gastric (stomach) cancer, childhood gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumors (gist) (soft tissue sarcoma), childhood gastrointestinalstromal tumors, germ cell tumors, childhood central nervous system germcell tumors, childhood extracranial germ cell tumors, extragonadal germcell tumors, ovarian germ cell tumors, testicular cancer, gestationaltrophoblastic disease, hairy cell leukemia, head and neck cancer, hearttumors, hepatocellular (liver) cancer, histiocytosis (Langerhans cellcancer), Hodgkin lymphoma, hypopharyngeal cancer (head and neck cancer),intraocular melanoma, childhood intraocular melanoma, islet cell tumors,(pancreatic neuroendocrine tumors), Kaposi sarcoma (soft tissuesarcoma), kidney (renal cell) cancer, Langerhans cell histiocytosis,laryngeal cancer (head and neck cancer), leukemia, lip and oral cavitycancer (head and neck cancer), liver cancer, lung cancer (non-small celland small cell), childhood lung cancer, lymphoma, male breast cancer,malignant fibrous histiocytoma of bone and osteosarcoma, melanoma,childhood melanoma, melanoma (intraocular eye), childhood intraocularmelanoma, Merkel cell carcinoma (skin cancer), mesothelioma, childhoodmesothelioma, metastatic cancer, metastatic squamous neck cancer withoccult primary (head and neck cancer), midline tract carcinoma with nutgene changes, mouth cancer (head and neck cancer), multiple endocrineneoplasia syndromes—see unusual cancers of childhood, multiplemyeloma/plasma cell neoplasms, mycosis fungoides (lymphoma),myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms,myelogenous leukemia, chronic (CML), myeloid leukemia, (acute AML),myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer(head and neck cancer), nasopharyngeal cancer (head and neck cancer),neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer, oralcancer (lip and oral cavity cancer and oropharyngeal cancer),osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer,childhood ovarian cancer, pancreatic cancer, childhood pancreaticcancer, pancreatic neuroendocrine tumors (islet cell tumors),papillomatosis, paraganglioma, childhood paraganglioma, paranasal sinusand nasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, childhood pheochromocytoma, pituitary tumor,plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma,pregnancy and breast cancer, primary central nervous system (CNS)lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer,recurrent cancer, renal cell (kidney) cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma, childhoodrhabdomyosarcoma (soft tissue sarcoma), childhood vascular tumors (softtissue sarcoma), Ewing sarcoma (bone cancer), Kaposi sarcoma (softtissue sarcoma), osteosarcoma (bone cancer), soft tissue sarcoma,uterine sarcoma, Sézary syndrome (lymphoma), skin cancer, childhood skincancer, small cell lung cancer, small intestine cancer, soft tissuesarcoma, squamous cell carcinoma of the skin, squamous neck cancer withoccult primary, stomach (gastric) cancer, childhood stomach, t-celllymphoma, testicular cancer, childhood testicular cancer, throat cancer,nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancerof the renal pelvis and ureter kidney (renal cell cancer), ureter andrenal pelvis (transitional cell cancer kidney renal cell cancer),urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginalcancer, childhood vaginal cancer, vascular tumors (soft tissue sarcoma),vulvar cancer, and Wilms tumor (and other childhood kidney tumors).

Coadministration w Anti-Cancer and Immunotherapy Agents

In another aspect, formulations and/or compounds provided herein arecoadministered or administered to an animal, subject or patient inconjunction with one or more chemotherapeutic compounds such asalkylating agents, antibodies and related agents with anti-tumorproperties, anthracyclines, antimetabolites, antitumor antibiotics,aromatase inhibitors, cytoskeletal disruptors (e.g. taxanes),epothilones, histone deacetylace inhibitors, kinase inhibitors,nucleoside analogues, topoisomerase inhibitors, retinoids, vincaalkaloids and derivatives, and the like. The administration orco-administration of one or more formulation or composition of theinvention and one or more chemotherapeutic agents can be used for thetreatment of tumors or cancer in an animal, subject or patient.

As an example, alkylating agents can be administered or coadministeredwith or as part of a formulation provided herein. Examples of analkylating agents that can be co-administered include mechlorethamine,chlorambucil, ifosfamide, melphalan, busulfan, carmustine, lomustine,procarbazine, dacardazine, cisplatin, carboplatin, mitomycin C,cyclophosphamide, ifosfamide, thiotepa, and dacarbazine, and analoguesthereof. See for example U.S. Pat. No. 3,046,301 describing thesynthesis of chlorambucil, U.S. Pat. No. 3,732,340 describing thesynthesis of ifosfamide, U.S. Pat. No. 3,018,302 for the synthesis ofcyclophosphamide, U.S. Pat. No. 3,032,584 describing the synthesis ofmelphalan, and Braunwald et al., “Harrison's Principles of InternalMedicine,” 15th Ed., McGraw-Hill, New York, N.Y., pp. 536-544 (2001) forclinical aspects of cyclophosphamide, chlorambucil, melphalan,ifosfamide, procarbazine, hexamethylmelamine, cisplatin, andcarboplatin. Examples of nucleoside analogues, include, but are notlimited to, fludarabine pentostatin, methotrexate, fluorouracil,fluorodeoxyuridine, CB3717, azacitidine, cytarabine, floxuridine,mercaptopurine, 6-thioguanine, cladribine, and analogues thereof.

In another aspect, formulations provided herein are administered withchemosensitising agents such as those described for example in U.S. Pat.No. 3,923,785 describing the synthesis of pentostatin, U.S. Pat. No.4,080,325 describing the synthesis of methotrexate, U.S. Pat. No.2,802,005 describing the synthesis of fluorouracil, and Braunwald etal., “Harrison's Principles of Internal Medicine,” 15th Ed.,McGraw-Hill, New York, N.Y., pp. 536-544 (2001) for clinical aspects ofmethotrexate, 5-fluorouracil, cytosine arabinoside, 6-mercaptopurine,6-thioguanine, and fludarabine phosphate. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992), incorporated by reference herein.

In another aspect, formulations provided herein can be administered orco-administered with diterpene compounds, including but not limited topaclitaxel, docetaxel, cabazitaxel, and the like.

In another aspect, formulations provided herein can be administered orco-administered with compounds that inhibit topoisomerase II orcompounds that otherwise interact with nucleic acids in cells. Suchcompounds include, for example, doxorubicin, epirubicin, etoposide,teniposide, mitoxantrone, and analogues thereof. In one example, thiscombination is used in treatment to reduce tumor cell contamination ofperipheral blood progenitor cells (PBSC) in conjunction with high-dosechemotherapy and autologous stem cell support (HDC-ASCT). See U.S. Pat.No. 6,586,428 to Geroni et al.

In another aspect, formulations provided herein can be administered orco-administered with immunotherapeutic agents. Immunotherapy has becomea promising approach to treat cancer. Kruger C., et al., Immune basedtherapies in cancer, Histol. Histopathol, 2007, v22, 687-696. The typesof immunotherapies used to treat cancer and can be categorized asactive, passive or hybrid (active and passive). Active immunotherapydirects the immune system to attack tumor cells by targeting TAAs.Passive immunotherapies enhance existing anti-tumor responses andinclude the use of checkpoint inhibitors, monoclonal antibodies,lymphocytes and cytokines. A suitable immunotherapeutic agent orimmunotherapy may be a biologic or biologically active agent such as anantibody or modified antibody or cell based therapy such as chimericantigen receptor therapy (CAR-T). It is recognized that there may beoverlap in categorizing and classifying such agent as biological agents,immunotherapeutic agents, cell-based therapeutics, biologicaltherapeutic agents and the like. Examples of approved antibodyimmunotherapeutics include, alemtuzumab, atezolizumab, avelumab,ipilimumab, durvalumab, nivolumab, ofatumumab, rituximab, andtrastuzumab. These and others are suitable for use in certainembodiments provided herein.

In another aspect, formulations can be administered or co-administeredwith biological therapeutic agents and other therapeutic drugs. Forexample, virulizin (Lorus Therapeutics), which is believed to stimulatethe release of tumor necrosis factor, TNF-α, by tumor cells in vitro andstimulate activation of macrophage cells. This can be used incombination with one or more formulation of the invention to increasecancer cell apoptosis and treat various types of cancers includingpancreatic cancer, malignant melanoma, kaposi's sarcoma (KS), lungcancer, breast cancer, uterine, ovarian and cervical cancer. Anotherexample is CpG 7909 (Coley Pharmaceutical Group), which is believed toactivate NK cells and monocytes and enhance ADCC. Cytokines such asinterferons and interleukins (e.g. EPO, thrombopoietin) are biologicalagents useful certain embodiments in combination with one or moreformulation of the invention. Other types of suitable biologicaltherapeutic agents include RNA and protein bases-agents such as enzymes.These therapeutic agents and others can also be used in combination withformulations provided herein.

Another example of a biological therapeutic agent that is used for thetreatment of certain cancers in certain embodiments are angiogensisinhibitors. Accordingly, formulations of the invention can also becombined with angiogensis inhibitors to increase anti-tumor effects.Angiogenisis is the growth of new blood vessels. This process allowstumors to grow and metastasize. Inhibiting angiogeneisis can helpprevent metastasis, and stop the spread of tumors cells. Angiogenisisinhibitors include, but are not limited to, angiostatin, endostatin,thrombospondin, platelet factor 4, Cartilage-derived inhibitor (CDI),retinoids, Interleukin-12, tissue inhibitor of metalloproteinase 1, 2and 3 (TIMP-1, TIMP-2, and TIMP-3) and proteins that block theangiogensis signaling cascade, such as anti-VEGF (Vascular EndothelialGrowth Factor) and IFN-alpha. Angiogenesis inhibitors can beadministered or co-administered with tumor specific constructs,including antigen-binding constructs capable of mediating, for example,ADCC and/or complement fixation or chemotherapy-conjugatedantigen-binding of the invention to combat various types of cancers, forexample, solid tumor cancers such as lung and breast cancer. Otherexamples of biological therapeutic agents include inhibitors ofE-cadherin and of epidermal growth factor receptor (EGFR). Knowninhibitors include erlotinib, an anti-integrin drug (Cilengitide),Cariporide, Eniporide and Amiloride.

In another aspect, formulations of the invention can be administered orco-administered with disease modifying anti-rheumatic agents (DMARagents) for the treatment of rheumatoid arthritis, psoriasis, ulcerativecolitus, systemic lupus erythematosus (SLE), Crohn's disease, ankylosingspondylitis, and various inflammatory disease processes. In suchtreatment, the constructs, for example, antigen-binding constructs, ofthe invention are commonly administered in conjunction with compoundssuch as azathioprine, cyclosporin, gold, hydroxychloroquine,methotrexate, penicallamine, sulphasalazine, and the like.

In another aspect, formulations provided herein can be used withpalliative (non-radical) operations to surgically remove tumors. In thisaspect, one or more formulations of the invention can be administeredbefore and after surgical extractions of tumors in order to reduce thelikelihood of metastasis and reoccurrence by killing any cancer cellsthat were not removed during the surgery.

Other diseases, conditions, and disorders described herein can betreated with formulations and methods provided herein.

Gout, Hyperuricaemia, Inflammatory Arthritis, and Related InflammatoryDisorders

Gout is a form of inflammatory arthritis characterized by recurrentattacks of a red, tender, hot, and swollen joint. Pain typically comeson rapidly in less than twelve hours. The joint at the base of the bigtoe is affected in about half of cases. It may also result in tophi,kidney stones, or urate nephropathy. Gout is due to persistentlyelevated levels of uric acid in the blood. This occurs due to acombination of diet and genetic factors. At high levels, uric acidcrystallizes and the crystals deposit in joints, tendons, andsurrounding tissues, resulting in an attack of gout. Gout occurs morecommonly in those who regularly eat meat or seafood, drink beer, or areoverweight. Diagnosis of gout may be confirmed by the presence ofcrystals in the joint fluid or in a deposit outside the joint. Blooduric acid levels may be normal during an attack. Disorders andconditions related to gout include, for example, osteoarthritis,rheumatoid arthritis and psoriatic arthritis.

In another aspect, methods of treating a subject, ameliorating, orpreventing a condition characterized by abnormal tissue levels of uricacid are provided. Methods of the invention may include the modulationof uric acid levels in subject. Accordingly, a method of treating adisease or disorder associated with abnormal levels of uric acid in apatient is provided comprising administering topically and/ortransdermally an effective amount of a pharmaceutical formulationcomprising one or more buffering agent to a patient having abnormallevels of uric acid and in need thereof, where the administration iseffective to treat or reduce the symptoms of abnormal levels of uricacid in said patient.

Certain embodiments provided herein are directed to method of treatinggout and related disorders. A method of treating gout and relateddisorders in accordance with the invention may comprise topically and/ortransdermally administering an effective amount of a formulationcomprising one or more buffering agent to a patient having gout and inneed thereof, wherein said administration is effective to treat orreduce the symptoms of gout in said patient.

Some embodiments are directed to a method of treating or amelioratinggout comprising topically and/or transdermally administering aneffective amount of a formulation comprising about 30% to about 35%sodium bicarbonate and, optionally, about at least 0.5% menthol to apatient having gout and in need thereof, wherein said administration iseffective to treat or reduce the symptoms of gout in said patient.

Some embodiments are directed to a method of treating or amelioratinggout comprising topically and/or transdermally administering aneffective amount of a formulation comprising about 30% to about 35%sodium bicarbonate and, optionally, about at least 0.5% menthol to apatient having gout and in need thereof, wherein said administration iseffective to treat or reduce the symptoms of gout in said patient.

In one embodiment, a method of treating or ameliorating gout is providedcomprising topically and/or transdermally administering an effectiveamount of a formulation comprising about 33% sodium bicarbonate andabout 0.5% menthol, to a patient having gout and in need thereof,wherein said administration is effective to treat or reduce the symptomsof gout in said patient.

In some embodiments, a condition disorder related to gout or directlyaffected or worsened by elevated levels of uric acid in the blood in asubject is treated, including for example a recurrent gout attack, goutyarthritis, hyperuricaemia, gout related cardiovascular disorders (e.g.hypertension, cardiovascular disease, coronary heart disease),Lesch-Nyhan syndrome, Kelley-Seegmiller syndrome, kidney disease, kidneystones, kidney failure, joint inflammation, inflammatory joint disease,arthritis, osteoarthritis, rheumatoid arthritis and psoriatic arthritis,urolithiasis, plumbism, hyperparathyroidism, psoriasis or sarcoidosis.

In certain embodiments, a method of treating gout and related disordersin accordance with the invention may also comprise topically and/ortransdermally administering an effective amount of a formulationcomprising one or more buffering agent to a patient having gout or arelated disorder and in need thereof, wherein said administration iseffective to treat or reduce the symptoms of gout in said patient, wherethe method further includes the administration or coadministration ofanother drug, therapeutic agent, or treatment. Suitable therapeuticagents for these embodiments include, for example, drugs for pain andinflammation in general (e.g. colchicine), corticosteroids, nonsteroidalanti-inflammatory drugs (NSAIDS), oral urate-lowering therapy (ULT, e.g.with xanthine oxidase inhibitors, drugs that lower uric acid (e.g.allopurinol, febuxostat, lesinurad, pegloticase, probenecid). In someembodiments, such therapeutic agents may be formulated and administeredas part of a formulation provided herein for topical and/or transdermaladministration.

Urinary and Renal Stones and Related Disorders

Kidney stones (renal lithiasis, nephrolithiasis) are common in humansand animals, and they typically comprise hard deposits made of mineralsand salts that form inside the bladder, kidneys, and urinary tract. Suchstones often form when the urine becomes concentrated, allowing mineralsto crystallize and stick together. Also, when a subject does not drinksufficient water there can be an accumulation of uric acid that isbelieved to be correlated with the formation of such stones. Anexcessively acidic environment in the urine of a subject is also thoughtto lead to the formation of kidney stones. They can be quite painful,and can lead to complications such as the blocking of the tubeconnecting the kidney to the bladder. Embodiments of the formulationsprovided herein have been found to be useful for the treatment, inhibit,amelioration of urinary and renal stones in a subject.

Accordingly, other embodiments provided herein are directed to methodsof urinary and renal stones and related disorders. In an exemplaryembodiment, a method of ameliorating or treating a urinary stone inaccordance with the invention typically comprises topically and/ortransdermally administering an effective amount of a pharmaceuticalformulation comprising one or more buffering agent to a patient having aurinary stone and in need thereof, wherein said administration iseffective to ameliorate, treat or reduce the symptoms of the urinarystone in said patient.

Examples of such conditions involving stones include, but not limited tobladder stones, kidney stones (calcium, calcium oxalate, calciumphosphate, cystine, magnesium ammonium phosphate, uric acid, struvite),renal stones, bilateral stone disease, urolithiasis during pregnancy,pediatric stones, stones in animals (e.g. urinary stones in animals),stones in patients with solitary kidneys, nephrolithiasis, other typesof stones (e.g. bladder, urinary), patients with bleeding diathesis andrelated disorders, urolithiasis, as well as in conjunction with medicalor surgical procedures such as a lithotripsy or ureteroscopy.

In certain embodiments, the patient is an animal such as a pet (e.g.cat, dog, bird), farm animal, or livestock. In non-limiting preferredembodiments, the urinary stone that is treated can be a bladder orkidney stone.

Skin Disorders

Other embodiments are directed to methods of treating a skin conditionor disorder in a patient. These embodiments typically comprise topicallyand/or transdermally administering an effective amount of a formulationcomprising one or more buffering agent to a patient having a skincondition or disorder and in need thereof, wherein said administrationis effective to ameliorate, treat or reduce the symptoms of the skincondition or disorder.

An exemplary but non-limiting skin disorder that is treated herein inparticular embodiments is melasma. Melasma is a common skin problem thatleads to skin pigmentation problems such as brown to gray-brown patches,usually on the face, cheeks, bridge of their nose, forehead, chin, andabove their upper lip.

Melasma is believed to be triggered or worsened by birth control pills,pregnancy, and hormone therapy, stress, thyroid disease, and sunexposure. Sun exposure is believed to cause melasma because ultravioletrays affect the cells that control pigment (melanocytes).

Thus, in certain embodiments methods of treating melasma are providedthat comprise topically and/or transdermally administering an effectiveamount of a formulation comprising one or more buffering agent to apatient having melasma and in need thereof, wherein said administrationis effective to ameliorate, treat or reduce the symptoms of the melasma.In some embodiments, methods of the invention use formulations providedherein in conjunction with or co-administered with another treatment formelasma (e.g. sun protection or a sun screen).

Another disorder or condition of the skin that is treated is skindamage. These embodiments typically comprise topically and/ortransdermally administering an effective amount of a formulationcomprising one or more buffering agent to a patient having skin damageand in need thereof, wherein said administration is effective toameliorate, treat or reduce the skin damage or symptoms associated withthe skin damage.

Other embodiments are directed to rejuvenating skin, and accordinglymethods of rejuvenating skin are provided that comprise topically and/ortransdermally administering an effective amount of a formulationcomprising one or more buffering agent to a subject in need of skinrejuvenation.

In certain embodiments, methods are provided that prevent or amelioratecollagen acylation in the skin of a patient. Alternative embodiments arealso directed to the pre-treatment of skin to prevent or ameliorate skindamage caused by collagen acylation and other factors.

Regulation of Na⁺/H⁺ Exchanger Isoform 1 (NHE1) Activity

In normal and neoplastic cells, the maintenance of pH homeostasis ischiefly regulated by the Na⁺/H⁺ exchanger isoform 1. Dysregulation ofNa⁺/H⁺ exchanger isoform 1 (NHE1) activity is the hallmark of cellsundergoing tumorigenesis and metastasis, the leading cause of patientmortality. While not being bound to any theory, the acidic tumormicroenvironment is thought to facilitate the development of resistanceto chemotherapy drugs and to promote extracellular matrix remodelingleading to metastasis. NHE1 activity has been shown to play an importantrole in the regulation of cell volume and shape, while also promotingcell growth, proliferation, differentiation, and apoptosis. It maycontribute to the development of multidrug resistance of tumor cells tomany chemotherapy drugs, which also hinders immune rejection of thesetumors. Weakly basic chemotherapy agents (e.g., doxorubicin) are likelypronated in acidic pH, which impedes their entrance into cells andprevents them from reaching their intracellular molecular targets.

It is another aspect of the invention to evaluate whether manipulatingthe tumor microenvironment through the modulation of NHE1 activity couldaid in chemotherapy treatment strategies in a co-adjuvant mannerpost-surgical intervention or, alternatively, in a co-neoadjuvant mannerprior to surgery.

A problem in the development of NHE1-specific inhibitors has been drivenby the need to counter the adverse effects of excessive exchangeractivity in the mammalian myocardium. Amiloride, a potassium-sparingdiuretic that has been used clinically, is a NHE inhibitor. Severalother drugs have since been and investigated in terms of their increasedselectivity and potency towards NHE1 inhibition and testing theseinhibitors for their anti-cancer properties is a subject of ongoingresearch. The two major families of these compounds are (i) the pyrazinederivatives (e.g., 5-(N,N-hexamethylene) amiloride),5-(N,N-dimethyl)amiloride, 5-(N-ethyl-N-isopropyl-amiloride)), and (ii)the benzoylguanidines (e.g., cariporide, eniporide, HOE-694).

Thus another aspect of the invention is to evaluate the inhibition ofNHE1 to be used as a target to increase the efficacy of anticancer drugs(e.g. chemotherapeutics, immunotherapeutic, biological agents describedherein) and as recent studies have lent credence to this hypothesis.

Also provided herein are methods of use of a NHE1 inhibitor administeredparenterally or topically directed against the various proteinsregulating the reversed pH gradient of tumors. The invention alsoenables the strategy of targeting NHE1 in synergistic combination with‘traditional’ pharmacological agents against one or more of itsup-stream activators. Both topical and parenteral administration thatare themselves non-systemic are contemplated as overcoming thedifficulties with oral administration, compromised first-pass metabolismand unacceptable bioavailability.

FIG. 16 shows the regulation of NHE1 and its roles in driving tumorbehaviors. A general scheme showing the major systems regulating theactivity of NHE1 with the resultant alkalinization of intracellular pH(pHi) and acidification of extracellular pH (pHe). These altered intra-and extra-cellular environments, in turn, drive a series of tumor cellbehaviors resulting in progression to more aggressive characteristics.

FIG. 17 illustrates the development of tumor metabolic microenvironment.General scheme showing how the dense, disorganized tumor interacts witha reduced circulatory availability to produce the tumor metabolicmicroenvironment, which is composed of low serum availability, hypoxiaand acidic extracellular pH. Exposure to this microenvironment furtherdrives metastatic progression.

Another aspect of the invention is directed to the use of NHE1inhibitors as an anticancer target. Due to the importance of NHE1 innumerous physiological and pathological processes, several inhibitorshave been developed. The inhibitors belong to two groups ofmodifications of the structure of the K-sparing diuretic, amiloride,(3.5-diamino-6-chloro-N-(diaminomethylene) pyrazinecarboxamide), thefirst compound found to have inhibitory activity.

The first series of other NHE1 inhibitory drugs based on the chemicalscaffold of amiloride had a slightly higher inhibitory activity andspecificity for NHE1. Later developed and also included are inhibitorswhere the pyrazine moiety of amiloride is substituted with a phenyl ringor a heterocycle pyridine to produce benzoylguanidines, including forexample HOE-694, cariporide (HOE-642), and eniporide (EMD85131). Thereis an additional series of NHE1 inhibitor compound whose structure isindependent of amiloride. One of this is SL-591227 which was the firstpotent and NHE1 selective non-guanidine inhibitor. The group of Tomodadeveloped phenoxazine derivatives (Phx-1) and Phx-3 that are highlyselective for NHE1 and which stimulate apoptosis in a variety of cancercell lines. Additionally, a

Also, researchers have synthesized a pyrimidine analog named compound 9thas been recently developed (Bristol-Meyers) that is reported to have avery high inhibitory activity, as much as 500-times more potent thancariporide, and much greater selectivity for NHE1 over NHE2 with a 52%oral bioavailability and a plasma half-life of 1.5 hours in rats. Itappears however that reports on the use of compound 9t either in vitroor in vivo are limited. However, compound 9t and other pyrimidineanalogs are contemplated as within the scope of certain embodiments.

Inhibitors of the amiloride series are also used in various embodiments,These include cariporide and eniporide.

Another aspect of the invention is to minimize the systemic dose of thedrug in order to dissociate the adverse effects and off-target effectsfrom the beneficial effects. Importantly, the potency of cariporide andsome other MHE inhibitors is related to the ionization state of theguanidine residues. Therefore, the acidic tumor microenvironment couldturn out to be an advantage in terms of dose-dependent side effects asthese compounds would be more efficient at inhibiting NHE1.

In certain embodiments combinations of NHE1 inhibitory drugs compoundscan be used. Such combinations can include both (i) cocktails ofinhibitors directed against the various proteins regulating the reversedpH gradient of tumors and (ii) the strategy of targeting NHE1 incombination with a ‘traditional’ pharmacological agent against one ormore of its up-stream activators (FIG. 16).

These strategies finally present the promise of a real paradigm shift incancer treatment towards manipulating the selective forces controllingthe dysregulated pH dynamics to reduce both the growth and themetastatic potential of tumors (FIG. 17).

Other aspects of the invention include the combination of protontransport inhibitors and the new ‘biological targeting’ of certaingrowth factor receptors to inhibit metastasis.

Other aspects include treating tumors with hyperthermia and there is agroup of studies showing that the lowering of pH by targeting NHE1 canstrongly enhance the thermosensitivity of the cancer cell. Theseembodiments have very real and important future possibilities for thecombined use of proton transporter inhibitors together withhyperthermia.

For these reasons, combination therapies are used herein to effectivelytreat many tumors screened for pertinent pathway dependence. In linewith this, the relatively high concentration of growth factors in tumorsand their positive role in NHE1 activation represents a perfect platformfor the topical administration of inhibitors through the combination theNHE1 inhibitors and the new biological targeting of some of these growthfactor-receptors. Accordingly, aspects of the invention include themulti-combined therapy of NHE1 inhibitors, such as cariporide, withinhibitors of one or more of these receptors having a role in bothactivating NHE1 and promoting tumor progression.

Other aspects include the parenterally or topically administeredcomposition as a stand alone or in synergistic combination withtraditional pharmacological agents. Topical administration is mostconveniently transdermal, but further includes transmembraneadministration, for example by suppository or intranasal application

Other aspects include the topical administration of agents and drugs,with or without occlusion in any manner and which are not conjugatedwith or delivered by means of penetration enhancing formulations, butare merely applied to the intact skin with or without massaging the skinfor the purpose of breaching the skin's permeation barrier.

The applicant surprisingly discovered that the combination of twohypothetical mechanisms, functioning in synergy, was successful intransdermal drug delivery (TDDD) of guest molecules of molecular weightsexceeding 500 Da and, in fact, beyond 150 kDa. These two synergisticmechanisms involve different interactions between the SPPs and thecellular moiety in the “transcellular” mechanism and the CPEs in the“extracellular” mechanism.

In another aspect, the invention discloses and provides integrative andcooperative methods with compositions that are directed to thesimultaneous and selective disruption of the cellular and lipid matrixcontributions to the SC permeation barrier in conjunction with thetransdermal delivery of agents. The mode of each physico-chemicalcomponent will be presented separately, although they may participatecooperatively in a chemical permeation enhancement (CPE) composition.

In another aspect a biochemical process, which is directed to thecellular component of the SC permeability barrier, is facilitated by asynergistic action of several biological processes, which combine toenhance transdermal drug delivery. In some embodiments, each of theseprocesses are used individually.

Other embodiments include the use of TD-1, as well as the other cationiccyclo-peptide variants identified as TDR-2, TDR-3 and TDR-7, in whicharginine substitutions are made at N-4, N-5 and N-7, and TDK-2, TDK-3and TDK-7, in which lysine substitutions are made at N-2, N-3 and N-7.Also embodied in this patent is cationic cyclo-peptide variant TD-34 asbis-substitute peptide in N-5 and N-6. The cyclic structure and thedisulfide constrained nature is critical for enhancement activity of thepeptides. The TDS series of the same amino acid sequence of cyclicstructure with TD-1 is further embodied as a modification viasubstitution of the N-terminal with three amino acids possessing thesame cationic group with various side-chain lengths. The enhancementactivity has been demonstrated to be proportional to side-chain lengthand identified as TDS-3>TDS-2>TDS-1.

While the exact mechanism is unclear, our studies have revealed theprofound activity of cell penetrating peptides (CPPs) with specialreference to TD-1, to be upon interactions with the skin cellularcomponents. The CPPs function by permeating through the transcellularroute passing through hydrophilic keratin-packed corneocytes that areembedded in multiple hydrophobic lipid bilayers. While partitioning intothe keratin-rich corneocytes, they form bridges that bind with thefilamentous keratin α-helices via hydrogen bonds in co-administration aspeptide-chaperones without interacting with the guest cargo or degradingthe lipid matrix. SPPs, in fact, enhance the lipid organization whilesimultaneously increasing skin electrical conductivity. TD-1 isnon-cytotoxic and non-irritating to skin.

It has been demonstrated that the CPPs also utilize the intercellularpathways via small gaps between the corneocytes by disruptingcell-to-cell junctional desmosomes expeditiously, thereby modifying thenormal ultrastructural spacing from about 30 nm to about 466 nm in aslittle as 30 minutes from topical administration. Transmission electronmicroscopy has revealed that the intercellular gaps are a transientprocess that will escort macromolecules across the SC permeation barrierrestoring the breaches in about one hour after application.

The co-administration of CPPs has been postulated to result in astatistically significant increase in percentage of α-helices ofkeratins, suggesting that CPPs stabilize these structural proteins(keratins). The intra-cellular keratins are stabilized by disulfidebonds, which are tightly packed either in α-chains (α-keratins) or inβ-sheet (β-keratins) structures. The high-degree of cross-linking by thedisulfide bonds, hydrophobic interactions and hydrogen bonds between thekeratin filament structures within the individual corneocytes confer itsmechanical stability preventing free drug transport.

Keratolytic agents will disrupt the tertiary structure and hydrogenbonds between individual keratin filaments, thereby promotingpenetration through intact skin. The administration of keratolyticagents will release keratin-bound active drug and enhancebioavailability.

One biochemical process is deployed to disrupt the disulfide linkage ofthe keratin filaments of which the corneocytes of the SC are comprised.This is contributed by means of a reducing agent containing a thiolmoiety. Thioglycolic Acid (TGA) @ 5% concentration is the preferredembodiment. Other agents, such as Dithiothretol (DTT), ß-Mercaptoethanol(ß-ME) and Urea Hydrogen Peroxide @ 17.5% concentration might besimilarly employed to act upon the hydrogen bonds, as well as thedisulfide bonds.

An additional keratolytic agent or enzyme, such as Proteinase K might beemployed to degrade the keratin substrate @ about 10 mg/mL The optimalpH of keratolytic activity is around pH 8, while activity is detected ina broad range of pH values between 6 to 11 for serine proteases.Chemical hydrolysis will further compromise the barrier propertycontributed by the corneocytes but the process is irreversible andconcentration-dependent.

The simultaneous application of the reducing agent has been demonstratedto have no adverse effect on the keratolytic enzymes and, in fact,allows the preferential access of the enzymes to the substrate forenhanced proteolytic attack.

Sigma-Aldrich offers an appropriate keratinolytic product (K4519-500UN),which is a non-specific serine protease with the capability of degradinginsoluble keratin substrates by cleaving non-terminal peptide bonds.

This patent further embodies an alternative to the reducingagent/keratolytic enzyme combination by means of two cooperating enzymesisolated from a keratin-degrading bacterium, Stenotrophomonas sp. strainD-1. These synergistic enzymes disrupt the disulfide bonds whilesimultaneously degrading the keratin substrate.

Formulations

A formulation for transdermal delivery may, for example, comprise twocomponents or it may comprise one or more buffering agent and apenetrant. Typically, however, a penetrant is less than 85% w/w. Theformulation may have a detergent of at least 1% w/w. For example, asuitable formulation may comprise about 10-56% w/w buffering agent and apenetrant. In one aspect, disclosed herein is a formulation fortransdermal delivery of one or more buffering agent through the skin ofa subject, comprising: a buffering agent comprising a carbonate salt inan amount between about 10-56% w/w; a penetrant portion in an amountbetween about 5 to 55% w/w; a detergent portion in an amount of at least1% w/w; and wherein the formulation comprises water in an amount fromnone up to about 77% w/w.

In another aspect, disclosed herein is a method for transdermal deliveryof a carbonate salt of the formulation comprising: a buffering agentcomprising a carbonate salt in an amount between about 10-45% w/w; apenetrant portion in an amount between about 5 to 55% w/w; a detergentportion in an amount between about 1 to 15% w/w; and wherein theformulation comprises water in an amount between about 15 to 65% w/w,through the skin of a subject, wherein the carbonate salt of theformulation is in an amount between about 15-32% w/w of the formulation.

In yet another aspect, disclosed herein is a formulation for transdermaldelivery of a therapeutic agent through the skin of a subject, whereinthe formulation comprises at least one active agent in an amounteffective for treatment of a condition in the subject and theformulation comprising: a buffering agent comprising a carbonate salt inan amount between about 10-45% w/w; a penetrant portion in an amountbetween about 5 to 55% w/w; a detergent portion in an amount betweenabout 1 to 15% w/w; wherein the formulation comprises water in an amountbetween about 15 to 65% w/w, through the skin of a subject, wherein thecarbonate salt of the formulation is in an amount between about 15-32%w/w of the formulation, therapeutic, and wherein the alkalinity of theformulation enhances penetration of the therapeutic agent.

In one aspect, disclosed herein is a formulation for transdermaldelivery of one or more buffering agent through the skin of a subject,comprising: a buffering agent comprising a carbonate salt in an amountbetween about 10-45% w/w; a penetrant portion in an amount between about5 to 55% w/w; a detergent portion in an amount between about 1 to 15%w/w; and wherein the formulation comprises water in an amount betweenabout 15 to 65% w/w, and wherein the formulation comprises less thanabout 12% w/w lecithin.

In another aspect, disclosed herein is a method for transdermal deliveryof a carbonate salt of the formulation comprising: a buffering agentcomprising a carbonate salt in an amount between about 10-45% w/w; apenetrant portion in an amount between about 5 to 55% w/w; a detergentportion in an amount between about 1 to 15% w/w; and wherein theformulation comprises water in an amount between about 15 to 65% w/w,and wherein the formulation comprises less than about 12% w/w lecithin,through the skin of a subject, wherein the carbonate salt of theformulation is in an amount between about 15-32% w/w of the formulation,wherein the formulation comprises less than about 12% w/w lecithin, andwherein the alkalinity of the formulation enhances penetration of thetherapeutic agent.

In yet another aspect, disclosed herein is a formulation for transdermaldelivery of a therapeutic agent through the skin of a subject, whereinthe formulation comprises at least one active agent in an amounteffective for treatment of a condition in the subject and theformulation comprising: a buffering agent comprising a carbonate salt inan amount between about 10-45% w/w; a penetrant portion in an amountbetween about 5 to 55% w/w; a detergent portion in an amount betweenabout 1 to 15% w/w; wherein the formulation comprises water in an amountbetween about 15 to 65% w/w, through the skin of a subject, wherein thecarbonate salt of the formulation is in an amount between about 15-32%w/w of the formulation, and wherein the formulation comprises less thanabout 12% w/w lecithin.

In some embodiments, a suitable formulation comprises: Lipmax™ in anamount between about 1-20% w/w; benzyl alcohol in an amount betweenabout 0.25 to 5% w/w; menthol in an amount between about 0.1-5% w/w;Pluronic® in an amount between about 0.1-5% w/w; water in an amountbetween about 10-80% w/w; sodium carbonate in an amount between about1-32% w/w; sodium bicarbonate in an amount between about 1-32% w/w;ethylene glycol tetraacetic acid in an amount less than about 5% w/w;propylene glycol in an amount between about 0.5-10% w/w; almond oil inan amount between about 0.5-10% w/w; cetyl alcohol in an amount betweenabout 0.5-10% w/w; lecithin in an amount less than about 12% w/w; CetiolUltimate® in an amount less than about 10% w/w; and ethanol in an amountbetween about 0.5-10% w/w.

In some embodiments, a suitable formulation comprises: Lipmax™ in anamount between about 1-20% w/w; benzyl alcohol in an amount betweenabout 0.25 to 5% w/w; menthol in an amount between about 0.1-5% w/w;Durasoft® in an amount between about 0.1-5% w/w; Pluronic® in an amountbetween about 0.1-5% w/w; water in an amount between about 10-80% w/w;sodium carbonate in an amount less than about 32% w/w; sodiumbicarbonate in an amount between about 1-32% w/w; ethylene glycoltetraacetic acid in an amount less than about 5% w/w; sodium decanoatein an amount less than about 5% w/w; propylene glycol in an amountbetween about 0.5-10% w/w; almond oil in an amount between about 0.5-10%w/w; zinc oxide in an amount less than about 2% w/w; cetyl alcohol in anamount between about 0.5-10% w/w; and ethanol in an amount between about0.5-10% w/w.

In some embodiments, a suitable formulation comprises: Water in anamount between about 10-80% w/w; Phospholipon® 90G in an amount betweenabout 0.5-16% w/w; Myritol® 312 in an amount between about 0.5-10% w/w;isopropyl palmitate in an amount between about 1-10% w/w; Cetiol®Ultimate in an amount between about 0.25-5% w/w; stearic acid in anamount between about 0.25-5% w/w; cetyl alcohol in an amount betweenabout 0.25-5% w/w; benzyl alcohol in an amount between about 0.25-5%w/w; propylene glycol in an amount between about 0.25-5% w/w; glycerinin an amount between about 0.25-5% w/w; ethanol in an amount betweenabout 0.25-5% w/w; Pluronic® in an amount between about 0.1-5% w/w;Lipmax™ in an amount between about 1-20% w/w; and sodium bicarbonate inan amount between about 1-32% w/w.

In some embodiments, a suitable formulation comprises: Siligel™ in anamount between about 1-5% w/w; water in an amount between about 10-80%w/w; Phospholipon® 90G in an amount between about 0.5-16% w/w; Myritol®312 in an amount between about 0.5-10% w/w; isopropyl palmitate in anamount between about 1-10% w/w; Cetiol® Ultimate in an amount betweenabout 0.25-5% w/w; stearic acid in an amount between about 0.25-5% w/w;cetyl alcohol in an amount between about 0.25-5% w/w; benzyl alcohol inan amount between about 0.25-5% w/w; propylene glycol in an amountbetween about 0.25-5% w/w; glycerin in an amount between about 0.25-5%w/w; ethanol in an amount between about 0.25-5% w/w; sodium hydroxide50% w/v in an amount between about 0.1-5% w/w; Lipmax™ in an amount lessthan about 20% w/w; and sodium bicarbonate in an amount between about1-32% w/w.

In some embodiments, a suitable formulation comprises: water in anamount between about 10-80% w/w; Phospholipon® 90G in an amount betweenabout 0.5-10% w/w; Myritol® 312 in an amount between about 0.5-10% w/w;isopropyl palmitate in an amount between about 0.5-10% w/w; Cetiol®Ultimate in an amount less than about 10% w/w; stearic Acid in an amountbetween about 0.25-5% w/w; cetyl alcohol in an amount between about0.25-5% w/w; benzyl alcohol in an amount between about 0.25-5% w/w;propylene glycol in an amount between about 0.25-5% w/w; glycerin in anamount between about 0.25-5% w/w; ethanol in an amount between about0.25-5% w/w; sodium hydroxide 50% w/v in an amount between about 0.1-5%w/w; and sodium bicarbonate in an amount between about 1-35% w/w.

In some embodiments, a suitable formulation comprises: water in anamount between about 10-40% w/w; Phospholipon® 90H in an amount betweenabout 0.5-20% w/w; Myritol® 312 in an amount between about 0.5-10% w/w;isopropyl palmitate in an amount between about 0.5-20% w/w; Cetiol®Ultimate in an amount less than about 10% w/w; stearic acid in an amountbetween about 0.25-5% w/w; cetyl alcohol in an amount between about0.25-5% w/w; benzyl alcohol in an amount between about 0.25-5% w/w;propylene glycol in an amount between about 0.25-5% w/w; glycerin in anamount between about 0.25-5% w/w; ethanol in an amount between about0.25-5% w/w; sodium hydroxide 50% w/v in an amount between about 0.1-5%w/w; and sodium bicarbonate in an amount between about 1-35% w/w.

In some embodiments, a suitable formulation comprises: water in anamount between about 10-40% w/w; Phospholipon® 90H in an amount betweenabout 0.5-20% w/w; Phospholipon® 90G in an amount between about 0.5-20%w/w; Myritol® 312 in an amount between about 0.5-10% w/w; isopropylpalmitate in an amount between about 0.5-20% w/w; Cetiol® Ultimate in anamount less than about 10% w/w; stearic acid in an amount between about0.25-5% w/w; cetyl alcohol in an amount between about 0.25-5% w/w;benzyl alcohol in an amount between about 0.25-5% w/w; propylene glycolin an amount between about 0.25-5% w/w; glycerin in an amount betweenabout 0.25-5% w/w; ethanol in an amount between about 0.25-5% w/w;sodium hydroxide 50% w/v in an amount between about 0.1-5% w/w; andsodium bicarbonate in an amount between about 1-35% w/w.

In some embodiments, a suitable formulation comprises: water in anamount between about 10-50% w/w; Pluronic® gel 30% in an amount betweenabout 5-30% w/w; isopropyl palmitate in an amount between about 0.5-20%w/w; stearic Acid in an amount between about 0.25-10% w/w; cetyl alcoholin an amount between about 0.25-10% w/w; benzyl alcohol in an amountbetween about 0.25-5% w/w; almond oil in an amount between about 0.5-10%w/w; propylene glycol in an amount between about 0.25-10% w/w; ethanolin an amount between about 0.25-5% w/w; sodium hydroxide 50% w/v in anamount between about 0.1-5% w/w; and sodium bicarbonate in an amountbetween about 1-32% w/w.

In some embodiments, a suitable formulation comprises: Siligel™ in anamount less than about 5% w/w; water in an amount between about 10-65%w/w; isopropyl palmitate in an amount between about 0.5-10% w/w; stearicAcid in an amount between about 0.25-10% w/w; cetyl alcohol in an amountbetween about 0.25-10% w/w; glycerin in an amount between about 0.25-5%w/w; Lipmax™ in an amount between about 0.25-10% w/w; ethanol in anamount less than about 5% w/w; benzyl alcohol in an amount less thanabout 5% w/w; sodium hydroxide 50% w/v in an amount between about 0.1-5%w/w; and sodium bicarbonate in an amount between about 1-32% w/w.

In some embodiments, a suitable formulation comprises: Aveeno® in anamount between about 20-85% w/w; and sodium bicarbonate (3DF) in anamount between about 15-45% w/w.

In some embodiments, a suitable formulation comprises: Aveeno® in anamount between about 20-85% w/w; and sodium bicarbonate (Milled #7) inan amount between about 15-45% w/w.

In some embodiments, a suitable formulation comprises: Siligel™ in anamount less than about 5% w/w; water in an amount between about 10-55%w/w; isopropyl palmitate in an amount between about 0.5-10% w/w; stearicAcid in an amount between about 0.25-5% w/w; Cetyl alcohol in an amountbetween about 0.25-10% w/w; almond oil in an amount between about0.5-10% w/w; propylene glycol in an amount between about 0.25-10% w/w;ethanol in an amount less than about 5% w/w; benzyl alcohol in an amountless than about 5% w/w; sodium hydroxide 50% w/v in an amount betweenabout 0.1-5% w/w; and sodium bicarbonate in an amount between about1-32% w/w.

The surprising effects achieved by the formulations and methods of thepresent invention are in part attributable to an improved formulationthat enhances delivery of a carbonate salt through the skin. In someembodiments, the formulation employs penetrants described US2009/0053290('290), WO2014/209910 ('910), and WO2017/127834. The presentformulations may include a nonionic surfactant. Applicant has found thatby employing carbonate salts with particle sizes as disclosed herein,delivered with the penetrants as disclosed herein, and in someembodiments providing a combination of a nonionic surfactant and a polargelling agent, the penetration capabilities of the carbonate salts ofthe resulting formulation and the effective level of delivery of thecarbonate salts has been enhanced. This enhanced level of penetrationwas also achieved using significantly less lecithin than anticipated ornone at all. This result was completely unexpected as it was believedthat relatively equal amounts of the benzyl alcohol and lecithinorganogel especially a somewhat higher concentration of benzyl alcoholthan lecithin organogel were responsible for the level of penetrationachieved by prior art formulations.

Briefly, the penetrants described in the above-referenced US and PCTapplications are based on combinations of synergistically actingcomponents. Many such penetrants are based on combinations of analcohol, such as benzyl alcohol to provide a concentration of 0.5-20%w/w of the final formulation with lecithin organogel present in thepenetrant to provide 25-70% w/w of the formulation. These penetrants arealso useful when the agent is a buffer, such as sodium bicarbonate, butless lecithin organogel may be required—e.g. less than 12% w/w when thesodium bicarbonate is present at high concentration as disclosed herein.

In some embodiments, the buffering component is any mildly basiccompound or combination that will result in a pH of 7-8 in themicroenvironment of the tumor cells. In some embodiments, theformulation has a pH of 7-10. Such buffers, in addition to carbonateand/or bicarbonate salts, include lysine buffers, chloroacetate buffers,tris buffers (i.e., buffers employing tris (hydroxymethyl) aminoethane),phosphate buffers and buffers employing non-natural amino acids withsimilar pKa values to lysine. In some embodiments, the carbonate and/orbicarbonate salt is in an amount between about 7-32 w/w of theformulation. For example, the enantiomers of native forms of such aminoacids or analogs of lysine with longer or shorter carbon chains orbranched forms thereof. Histidine buffers may also be used. Typically,the concentration of buffer in the compositions is in the range of10-50% w/w. More typical ranges for sodium bicarbonate or sodiumcarbonate or both are 10-35% by weight. In some embodiments, thecarbonate salt is in an amount between about 15-32% w/w of theformulation.

Alternatively, the penetrant component comprises a completion componentas well as one or more electrolytes sufficient to impart viscosity andviscoelasticity, one or more surfactants and an alcohol. The completioncomponent can be a polar liquid, a non-polar liquid or an amphiphilicsubstance.

The percentage of carbonate salt in the formulation will depend upon theamount required to be delivered in order to have a useful effect ontreating the disorder. In general, the carbonate salt may be present inthe formulation in an amount as low as 1% w/w up to about 50% w/w.Typical concentrations may include 15-32% w/w. Since the requiredpercentage of carbonate salt depends on the frequency of administration,as well as the time allotted for administration for each application,the level of carbonate salt may be varied over a wide range. In someembodiments, the carbonate salt is sodium carbonate and/or sodiumbicarbonate milled to a particle size is less than 200 μm. In someembodiments, the carbonate salt is sodium carbonate and/or sodiumbicarbonate milled to a particle size is less than 70 μm. In someembodiments, the carbonate salt is sodium carbonate and/or sodiumbicarbonate milled to a particle size is less than 70 μm, wherein thesodium bicarbonate is solubilized in the formulation in an amount lessthan 20% w/w of the formulation. In some embodiments, the carbonate saltis sodium carbonate and/or sodium bicarbonate milled to a particle sizeis less than 70 μm, wherein particle sizes less than about 10 μm have anenhanced penetration thru the skin of a subject. In some embodiments,the sodium carbonate and/or sodium bicarbonate are jet milled to aparticle size less than about 70 μm. In some embodiments, the sodiumbicarbonate is Sodium Bicarbonate USP Grade 3DF that has a particle sizedistribution less than 70 μm.

The formulations of the disclosure may be prepared in a number of ways.Typically, the components of the formulation are simply mixed togetherin the required amounts. However, it is also desirable in some instancesto, for example, carry out dissolution of a carbonate salt and then adda separate preparation containing the components aiding the delivery ofthe carbonate salts in the form of a carrier. The concentrations ofthese components in the carrier, then, will be somewhat higher than theconcentrations required in the final formulation. Thus, sodiumbicarbonate may first be dissolved in water and then added to a carriercomprising an alcohol, lecithin and optionally a combination of anonionic surfactant and polar gelling agent, or of ionic detergent.Alternatively, some subset of these components can first be mixed andthen “topped off” with the remaining components either simultaneously orsequentially. The precise manner of preparing the formulation willdepend on the choice of carbonates and the percentages of the remainingcomponents that are desirable with respect to that carbonate salt. Insome embodiments, the water is in an amount between about 10-85% w/w,15-50% w/w, or 15-45% w/w of the formulation.

The penetrant portion is a multi-component mixture, whereby theparticular concentrations of the penetration enhancers are informed inpart by the molecular mass of the sodium bicarbonate, or sodiumbicarbonate and the therapeutic agent to be transported. The formulationenables the sodium bicarbonate and/or therapeutic agent to becomebio-available to the target site within minutes of topicaladministration. The formulations permit the use of minimalconcentrations of therapeutic agents, as little as. 1/1000th ofconcentrations required of alternative processes, while enablingbioactivity and positive clinical outcomes simultaneously. In someembodiments, the penetrant portion comprises an alcohol in an amountless than 5% w/w of the formulation.

One important aspect of the invention is based on the above-notedrecognition that some tumors do not respond to buffer treatment as theirmicroenvironment is not acidic and at least some of these tumors achievemetastasis by elevation of certain proteolytic enzymes that break downthe extracellular matrix (ECM). If buffer treatment is contemplated,tumor cells from the biopsy of a solid tumor in a subject are thereforepreferably cultured and tested in advance of treatment to insureresponsiveness to buffer. Such evaluation can be carried out by anysuitable means, including measurement of pH, assessment of the levels ofrelevant proteases, and invasion assays as impacted by buffer treatmentas described in Bailey, K. M. et al (2014) supra. One important suchassay is a glycolytic stress assay as described therein. Cell culturesof biopsied tumors that appear not to respond to buffer treatment asshown by such assays may benefit from administration of otherantimetastatic agents and inclusion of such agents in the compositionsof the invention that include buffers would also be beneficial. Thus,treatment with buffer-containing compositions alone may becontraindicated and the subject is not administered buffer as the soleactive agent but diverted to alternative treatment. This does not mean,of course, that buffer is necessarily omitted from formulations used toadminister alternative active agents.

The formulations comprise mixtures wherein the components interactsynergistically and induce skin permeation enhancements better than thatinduced by the individual components. Synergies between chemicals can beexploited to design potent permeation enhancers that overcome theefficacy limitations of single enhancers. Several embodiments disclosedherein utilize three to five distinct permeation enhancers.

For topical administration, and in particular transdermaladministration, the formulation will comprise penetrants includingeither or both chemical penetrants (CPEs) and peptide-based cellularpenetrating agents (CPPs) that encourage transmission across the dermisand/or across membranes including cell membranes, as would be the casein particular for administration by suppository or intranasaladministration, but for transdermal administration as well. Particularlysuitable penetrants especially for those that contain at least one agentother than buffer include those that are described in theabove-referenced US2009/0053290 ('290), WO2014/209910 ('910), andWO2017/127834. In addition to formulations with penetrants, transdermaldelivery can be affected by mechanically disrupting the surface of theskin to encourage penetration, or simply by supplying the formulationapplied to the skin under an occlusive patch.

Alternatively, the penetrant portion comprises a completion component aswell as one or more electrolytes sufficient to impart viscosity andviscoelasticity, one or more surfactants and an alcohol. The completioncomponent can be a polar liquid, a non-polar liquid or an amphiphilicsubstance. The penetrant may further comprise a keratinolytic agenteffective to reduce thiol linkages, disrupt hydrogen bonding and/oreffect keratin lysis and/or a cell penetrating peptide (sometimesreferred to as a skin-penetrating peptide) and/or a permeation enhancer.

Lecithin organogel is a combination of lecithin with a gellingcomponent, which is typically amphiphilic. Suitable gelling componentsalso include isopropyl palmitate, ethyl laurate, ethyl myristate andisopropyl myristate. In some embodiments, the formulation comprises agelling agent in an amount less than 5% w/w of the formulation. Certainhydrocarbons, such as cyclopentane, cyclooctane, trans-decalin,trans-pinane, n-pentane, n-hexane, n-hexadecane may also be used. Thus,an important permeation agent is a lecithin organogel, wherein thecombination resulting from lecithin and the organic solvent acts as apermeation agent. In some embodiments, the penetrant portion compriseslecithin organogel, an alcohol, a surfactant, and a polar solvent. Insome embodiments, the lecithin organogel is a combination of soylecithin and isopropyl palmitate. In some embodiments, the penetrantportion comprises lecithin and isopropyl palmitate, undecane,isododecane, isopropyl stearate, or a combination thereof. In someembodiments, the formulation comprises Lipmax™ (sold by Lucas MeyerCosmetics) in an amount between about 1-20% w/w or an equivalent 50/50mixture of isopropyl palmitate and lecithin. Lecithin organogels areclear, thermodynamically stable, viscoelastic, and biocompatiblejelly-like phases composed of hydrated phospholipids and appropriateorganic liquid. An example of a suitable lecithin organogel is lecithinisopropyl palmitate, which is formed when isopropyl palmitate is used todissolve lecithin. The ratio of lecithin to isopropyl palmitate may be50:50. Illustrated below in the Examples is a formulation containing soylecithin in combination with isopropyl palmitate; however, otherlecithins could also be used such as egg lecithin or syntheticlecithins. Various esters of long chain fatty acids may also beincluded. Methods for making such lecithin organogels are well known inthe art. In most embodiments, the lecithin organogel is present in thefinal formulation is less than about 20% w/w. In those compositions usedto dissolve fat deposits, to alleviate pain from fat removal or inanhydrous compositions, the concentration of lecithin organogel may beas low as 0.5% w/w, 1% w/w, 5% w/w, 10% w/w or 20% w/w. In someembodiments, the penetrant portion comprises a mixture of xanthan gum,lecithin, sclerotium gum, pullulan, or a combination thereof in anamount less than 2% w/w, 5% w/w, or 10% w/w of the formulation. In someembodiments, the formulation comprises Siligel™ in an amount betweenabout 1-5 w/w or 5-15% w/w, or an equivalent mixture of xanthan gum,lecithin, sclerotium gum, and pullulan. In some embodiments, thepenetrant portion comprises a mixture of caprylic triglycerides andcapric triglycerides in amount less than 2% w/w, 8% w/w, or 10% w/w ofthe formulation. In some embodiments, the formulation comprises Myritol®312 in an amount between about 0.5-10% w/w, or an equivalent mixture ofcaprylic triglycerides and capric triglycerides.

In some embodiments, the penetrant portion comprises phosphatidylcholine in amount less than 12% w/w or 18% w/w of the formulation. Insome embodiments, the penetrant portion comprises a phospholipid inamount less than 12% w/w or 18% w/w of the formulation. In someembodiments, the penetrant portion comprises a mixture of tridecane andundecane in amount less than 2% w/w, 5% w/w, or 8% w/w of theformulation. In some embodiments, the formulation comprises CetiolUltimate® in an amount less than about 2% w/w, 5% w/w, or 10% w/w, or anequivalent mixture of tridecane and undecane. In some embodiments, thepenetrant portion comprises cetyl alcohol in amount less than 2% w/w, 5%w/w, or 8% w/w of the formulation. In some embodiments, the penetrantportion comprises benzyl alcohol in an amount less than about 2% w/w, 5%w/w, or 8% w/w. In some embodiments, the penetrant portion comprisesstearic acid in an amount less than 2% w/w, 5% w/w, or 8% w/w of theformulation.

Lecithin organogels may be in the form of vesicles, microemulsions andmicellar systems. In the form of self-assembled structures, such asvesicles or micelles, they can fuse with the lipid bilayers of thestratum corneum, thereby enhancing partitioning of encapsulated drug, aswell as a disruption of the ordered bilayers structure. An example of aphospholipid-based permeation enhancement agent comprises amicro-emulsion-based organic gel defined as a semi-solid formationhaving an external solvent phase immobilized within the spaces availableof a three-dimensional networked structure. This micro-emulsion-basedorganic gel in liquid phase is characterized by1,2-diacyl-sn-glycero-3-phosphatidyl choline, and an organic solvent,which is at least one of: ethyl laureate, ethyl myristate, isopropylmyristate, isopropyl palmitate; cyclopentane, cyclooctane,trans-decalin, trans-pinane, n-pentane, n-hexane, n-hexadecane, andtripropylamine.

The lecithin organogels are formulated with an additional component toassist in the formation of micelles or vascular structures. In oneapproach, the organogels are formulated with a polar component such aswater, glycerol, ethyleneglycol or formamide, in particular with water.In general, a nonionic detergent such as a poloxamer in aqueous solutionis used to top off. Alternatively, an anhydrous composition may beobtained by using, instead of a polar component, a material such as abile salt. When formulated with bile salts, the mi cellular nature ofthe composition is altered so that rather than a more or less sphericalvesicular form, the vesicles become wormlike and are able to accommodatelarger guest molecules, as well as penetrate the epidermis moreeffectively. Suitable bile salts include salts of deoxycholic acid,taurocholic acid, glycocholic acid, taurochenodeoxycholic acid,glycochenodeoxycholic acid, cholic acid and the like. Certaindetergents, such as Tween® 80 or Span® 80 may be used as alternatives.The percentage of these components in the anhydrous forms of thecomposition is in the range of 1% w/w-15% w/w. In some embodiments, therange of bile salt content is 2%-6% w/w or 1%-3.5% w/w. In theseessentially anhydrous forms, powdered or micronized nonionic detergentis used to top off, typically in amounts of 20%-60% w/w. In one approachto determine the amount of bile salt, the % is calculated by dividingthe % w/w of lecithin by 10.

An additional component in the formulations of the disclosure is analcohol. Benzyl alcohol and ethanol are illustrated in the Examples. inparticular, derivatives of benzyl alcohol which contain substituents onthe benzene ring, such as halo, alkyl and the like. The weightpercentage of benzyl or other related alcohol in the final compositionis 0.5-20% w/w, and again, intervening percentages such as 1% w/w, 2%w/w, 5% w/w, 7% w/w, 10% w/w, and other intermediate weight percentagesare incl tided. Due to the aromatic group present in a permeationenhancement formulation such as benzyl alcohol, the molecule has a polarend (the alcohol end) and a non-polar end (the benzene end). Thisenables the agent to dissolve a wider variety of drugs and agents. Thealcohol concentration is substantially lower than the concentration ofthe lecithin organogel in the composition.

In some embodiments, as noted above, the performance of the formulationsis further improved by including a nonionic detergent and polar gellingagent or including bile salts and a powdered surfactant. In both aqueousand anhydrous forms of the composition, detergents, typically nonionicdetergents are added. In general, the nonionic detergent should bepresent in an amount of at least 2% w/w to 60% w/w. Typically, in thecompositions wherein the formulation is topped off with a polar oraqueous solution containing detergent, the amount of detergent isrelatively low—e.g., 2%-25% w/w, or 5-15% w/w or 7-12% w/w. However, incompositions comprising bile salts that are essentially anhydrous andare topped-off by powdered detergent, relatively higher percentages areusually used—e.g., 20%-60% w/w.

In some embodiments, the nonionic detergent provides suitable handlingproperties whereby the formulations are gel-like or creams at roomtemperature. To exert this effect, the detergent, typically a poloxamer,is present in an amount between about 2-12% w/w, preferably betweenabout 5-25% w/w in polar formulations. In the anhydrous forms of thecompositions, the detergent is added in powdered or micronized form tobring the composition to 100% and higher amounts are used. Incompositions with polar constituents, rather than bile salts, thenonionic detergent is added as a solution to bring the composition to100%. If smaller amounts of detergent solutions are needed due to highlevels of the remaining components, more concentrated solutions of thenonionic detergent are employed. Thus, for example, the percentdetergent in the solution may be 10% to 40% or 20% or 30% andintermediate values depending on the percentages of the othercomponents.

Suitable nonionic detergents include poloxamers such as Pluronic® andany other surfactant characterized by a combination of hydrophilic andhydrophobic moieties. Poloxamers are triblock copolymers of a centralhydrophobic chain of polyoxypropylene flanked by two hydrophilic chainsof polyethyleneoxide. Other nonionic surfactants include long chainalcohols and copolymers of hydrophilic and hydrophobic monomers whereblocks of hydrophilic and hydrophobic portions are used.

In some embodiments, the formulation also contains surfactant,typically, nonionic surfactant at 2-25% w/w along with a polar solventwherein the polar solvent is present in an amount at least in molarexcess of the nonionic surfactant. In these embodiments, typically, thecomposition comprises the above-referenced amounts of lecithin organogeland benzyl alcohol along with a carbonate salt with a sufficient amountof a polar solution, typically an aqueous solution or polyethyleneglycol solution that itself contains 10%-40% of surfactant, typicallynonionic surfactant to bring the composition to 100%.

Other examples of surfactants include polyoxyethylated castor oilderivatives such as HCO-60 surfactant sold by the HallStar Company;nonoxynol; octoxynol; phenylsulfonate; poloxamers such as those sold byBASF as Pluronic® F68, Pluronic® F127, and Pluronic® L62; polyoleates;Rewopal® HVIO, sodium laurate, sodium lauryl sulfate (sodium dodecylsulfate); sodium oleate; sorbitan dilaurate; sorbitan dioleate; sorbitanmonolaurate such as Span® 20 sold by Sigma-Aldrich; sorbitanmonooleates; sorbitan trilaurate; sorbitan trioleate; sorbitanmonopalmitate such as Span® 40 sold by Sigma-Aldrich; sorbitan stearatesuch as Span® 85 sold by Sigma-Aldrich; polyethylene glycol nonylphenylether such as Synperonic® NP sold by Sigma-Aldrich;p-(1,1,3,3-tetramethylbutyl)-phenyl ether sold as Triton™ X-100 sold bySigma-Aldrich; and polysorbates such as polyoxyethylene (20) sorbitanmonolaurate sold as Tween® 20, polysorbate 40 (polyoxyethylene (20)sorbitan monopalmitate) sold as Tween® 40, polysorbate 60(polyoxyethylene (20) sorbitan monostearate) sold as Tween® 60,polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) sold as Tween®80, and polyoxyethylenesorbitan trioleate sold as Tween® 85 bySigma-Aldrich. The weight percentage range of nonionic surfactant is inthe range of 3% w/w-15% w/w, and again includes intermediate percentagessuch as 5% w/w, 7% w/w, 10% w/w, 12% w/w, and the like. In someembodiments, the detergent portion comprises a nonionic surfactant in anamount between about 2-25% w/w of the formulation; and a polar solventin an amount less than 5% w/w of the formulation. In some embodiments,the nonionic surfactant is a poloxamer and the polar solvent is water,an alcohol, or a combination thereof. In some embodiments, the detergentportion comprises poloxamer, propylene glycol, glycerin, ethanol, 50%w/v sodium hydroxide solution, or a combination thereof. In someembodiments, the detergent portion comprises glycerin in an amount lessthan 3% w/w of the formulation.

In the presence of a polar gelling agent, such as water, glycerol,ethyleneglycol or formamide, a micellular structure is also oftenachieved. Typically, the polar agent is in molar excess of the nonionicdetergent. The inclusion of the nonionic detergent/polar gelling agentcombination results in a more viscous and cream-like or gel-likeformulation which is suitable for application directly to the skin. Thisis typical of the aqueous forms of the composition.

In some embodiments other additives are included such as a gellingagent, a dispersing agent and a preservative. An example of a suitablegelling agent is hydroxypropylcellulose, which is generally available ingrades from viscosities of from about 5 cps to about 25,000 cps such asabout 1500 cps. All viscosity measurements are assumed to be made atroom temperature unless otherwise stated. The concentration ofhydroxypropylcellulose may range from about 1% w/w to about 2% w/w ofthe composition. Other gelling agents are known in the art and can beused in place of, or in addition to hydroxypropylcellulose. An exampleof a suitable dispersing agent is glycerin. Glycerin is typicallyincluded at a concentration from about 5% w/w to about 25% w/w of thecomposition. A preservative may be included at a concentration effectiveto inhibit microbial growth, ultraviolet light and/or oxygen-inducedbreakdown of composition components, and the like. When a preservativeis included, it may range in concentration from about 0.01% w/w to about1.5% w/w of the composition.

Typical components that may also be included in the formulations arefatty acids, terpenes, lipids, and cationic, and anionic detergents. Insome embodiments, the formulation further comprises tranexamic acid inan amount less than 2% w/w, 5% w/w, or 10% w/w of the formulation. Insome embodiments, the formulation further comprises a polar solvent inan amount less than 2% w/w, 5% w/w, 10% w/w, or 20% w/w of theformulation. In some embodiments, the formulation further comprises ahumectant, an emulsifier, an emollient, or a combination thereof. Insome embodiments, the formulation further comprises ethylene glycoltetraacetic acid in an amount less than about 2% w/w, 5% w/w, or 10%w/w. In some embodiments, the formulation further comprises almond oilin an amount less than about 5% w/w. In some embodiments, theformulation further comprises a mixture of thermoplastic polyurethaneand polycarbonate in an amount less than about 5% w/w. In someembodiments, the formulation further comprises phosphatidylethanolaminein an amount less than about 5 w/w. In some embodiments, the formulationfurther comprises an inositol phosphatide in an amount less than about5% w/w.

Other solvents and related compounds that may be used in someembodiments include acetamide and derivatives, acetone, n-alkanes (chainlength between 7 and 16), alkanols, diols, short chain fatty acids,cyclohexyl-1,1-dimethylethanol, dimethyl acetamide, dimethyl formamide,ethanol, ethanol/d-limonene combination, 2-ethyl-1,3-hexanediol,ethoxydiglycol (Transcutol® by Gattefosse, Lyon, France), glycerol,glycols, lauryl chloride, limonene N-methylformamide, 2-phenylethanol,3-phenyl-1-propanol, 3-phenyl-2-propen-1-ol, polyethylene glycol,polyoxyethylene sorbitan monoesters, polypropylene glycol 425, primaryalcohols (tridecanol), 1,2-propane diol, butanediol, C₃-C₆ triols ortheir mixtures and a polar lipid compound selected from C₁₆ or C₁₈monounsaturated alcohol, C₁₆ or C₁₈ branched saturated alcohol and theirmixtures, propylene glycol, sorbitan monolaurate sold as Span® 20 bySigma-Aldrich, squalene, triacetin, trichloroethanol, trifluoroethanol,trimethylene glycol and xylene.

Fatty alcohols, fatty acids, fatty esters, are bilayer fluidizers thatmay be used in some embodiments. Examples of suitable fatty alcoholsinclude aliphatic alcohols, decanol, lauryl alcohol (dodecanol),unolenyl alcohol, nerolidol, 1-nonanol, n-octanol, and oleyl alcohol.Examples of suitable fatty acid esters include butyl acetate, cetyllactate, decyl N,N-dimethylamino acetate, decyl N,N-dimethylaminoisopropionate, diethyleneglycol oleate, diethyl sebacate, diethylsuccinate, diisopropyl sebacate, dodecyl N,N-dimethyamino acetate,dodecyl (N,N-dimethylamino)-butyrate, dodecyl N,N-dimethylaminoisopropionate, dodecyl 2-(dimethyamino) propionate, E0-5-oleyl ether,ethyl acetate, ethylaceto acetate, ethyl propionate, glycerolmonoethers, glycerol monolaurate, glycerol monooleate, glycerolmonolinoleate, isopropyl isostearate, isopropyl linoleate, isopropylmyristate, isopropyl myristate/fatty acid monoglyceride combination,isopropyl palmitate, methyl acetate, methyl caprate, methyl laurate,methyl propionate, methyl valerate, 1-monocaproyl glycerol,monoglycerides (medium chain length), nicotinic esters (benzyl), octylacetate, octyl N,N-dimethylamino acetate, oleyl oleate, n-pentylN-acetylprolinate, propylene glycol monolaurate, sorbitan dilaurate,sorbitan dioleate, sorbitan monolaurate, sorbitan monolaurate, sorbitantrilaurate, sorbitan trioleate, sucrose coconut fatty ester mixtures,sucrose monolaurate, sucrose monooleate, tetradecyl N,N-dimethylaminoacetate. Examples of suitable fatty acid include alkanoic acids, capridacid, diacid, ethyloctadecanoic acid, hexanoic acid, lactic acid, lauricacid, linoelaidic acid, linoleic acid, linolenic acid, neodecanoic acid,oleic acid, palmitic acid, pelargonic acid, propionic acid, and vaccenicacid. Examples of suitable fatty alcohol ethers include α-monoglycerylether, E0-2-oleyl ether, E0-5-oleyl ether, E0-10-oleyl ether, etherderivatives of polyglycerols and alcohols, and(1-O-dodecyl-3-O-methyl-2-O-(2′,3′-dihydroxypropyl glycerol).

Examples of completing agents that may be used in some embodimentsinclude β- and γ-cyclodextrin complexes, hydroxypropyl methylcellulose(e.g., Carbopol® 934), liposomes, naphthalene diamide diimide, andnaphthalene diester diimide.

One or more anti-oxidants may be included, such as vitamin C, vitamin E,proanthocyanidin and α-lipoic acid typically in concentrations of0.1%-2.5% w/w.

In some applications, it is desirable to adjust the pH of theformulation to assist in permeation or to adjust the nature of thecarbonate and/or of the target compounds in the subject. In someinstances, the pH is adjusted to a level of pH 9-11 or 10-11 which canbe done by providing appropriate buffers or simply adjusting the pH withbase.

In some applications, in particular when the therapeutic agent includesan anesthetic, epinephrine or an alternate vasoconstrictor, such asphenylephrine or epinephrine sulfate may be included in the formulationif a stabilizing agent is present. Otherwise, the epinephrine should beadministered in tandem since epinephrine is not stable at alkali pH.

In any of the anesthetic compositions, it may be desirable to administerthe epinephrine in tandem with the transdermal anesthetic.Alternatively, treatment of the epinephrine with a chelator, such as theiron chelator Desferal® may stabilize the epinephrine sufficiently toinclude it in the transdermal formulation.

It is understood that some tumors do not respond to treatment withbuffer, but apparently metastasize by virtue of elevated levels ofproteases that attack the extracellular matrix surrounding the tumor. Inany event, breakdown of the ECM would encourage metastasis. Therefore,an additional active agent that is optionally included in thecompositions of the invention is one or more appropriate proteaseinhibitors. Particularly important are inhibitors of cathepsins, forexample of cathepsin B, and inhibitors of matrix metalloproteinases(MMPs). These components are active alone or augment the effect ofbuffer for tumors that are not resistant to buffer treatment.

The formulations may include other components that act as excipients orserve purposes other than active anti-tumor effects. For example,preservatives like antioxidants e.g., ascorbic acid or α-lipoic acid andantibacterial agents may be included. Other components apart fromtherapeutically active ingredients and components that are the primaryeffectors of dermal penetration may include those provided for aestheticpurposes such as menthol or other aromatics, and components that affectthe physical state of the composition such as emulsifiers, for example,Durasoft® (which is a mixture of thermoplastic polyurethane andpolycarbonate). Typically, these ingredients are present in very smallpercentages of the compositions. It is understood that these latterancillary agents are neither therapeutically ingredients nor are theycomponents that are primarily responsible for penetration of the skin.The components that primarily effect skin penetration have been detailedas described above. However, some of these substances have somecapability for effecting skin penetration. See, for example, Kunta, J.R. et al, J. Pharm. Sci. (1997) 86:1369-1373, describing penetrationproperties of menthol.

In embodiments where a bile salt is added to the combination of benzylalcohol and lecithin organogel in lieu of topping off with an aqueousmedium, micelles that would have been relatively spherical may becomeelongated and worm-like thus permitting superior penetration of thestratum corneum of the epidermis. The worm like formation of themicelles is particularly helpful in accommodating higher molecularweight therapeutic agents. As is known, bile salts are facialamphiphiles and include salts of taurocholic acid, glycocholic acid,taurochenodeoxycholic acid, glycochenodeoxycholic acid, cholic acid,deoxycholic acid. Detergents are also useful in lieu of bile salts andinclude Tween® 80 and Span® 80.

In another aspect, certain embodiments are directed to a sustainedrelease drug delivery platform releases a therapeutic compound orcompounds disclosed and made as a formulation described herein over aperiod of, without limitation, about 3 days after administration, about7 days after administration, about 10 days after administration, about15 days after administration, about 20 days after administration, about25 days after administration, about 30 days after administration, about45 days after administration, about 60 days after administration, about75 days after administration, or about 90 days after administration. Inother aspects of this embodiment, a sustained release drug deliveryplatform releases a therapeutic compound or compounds disclosed hereinwith substantially first order release kinetics over a period of,without limitation, at least 3 days after administration, at least 7days after administration, at least 10 days after administration, atleast 15 days after administration, at least 20 days afteradministration, at least 25 days after administration, at least 30 daysafter administration, at least 45 days after administration, at least 60days after administration, at least 75 days after administration, or atleast 90 days after administration.

The formulation described in this specification may also comprise morethan one therapeutic compound as desired for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect the other proteins. The formulations to be used forin vivo administration can be sterile. This can be accomplished, forinstance, without limitation, by filtration through sterile filtrationmembranes, prior to, or following, preparation of the formulation orother methods known in the art, including without limitation,pasteurization.

Packaging and instruments for administration may be determined by avariety of considerations, such as, without limitation, the volume ofmaterial to be administered, the conditions for storage, whether skilledhealthcare practitioners will administer or patient self-compliance, thedosage regime, the geopolitical environment (e.g., exposure to extremeconditions of temperature for developing nations), and other practicalconsiderations.

In certain embodiments, kits can comprise, without limitation, one ormore cream or lotion comprising one or more formulations describedherein. In various embodiments, the kit can comprise formulationcomponents for transdermal, topical, or subcutaneous administration,formulated to be administered as an emulsion coated patch. In all ofthese embodiments and others, the kits can contain one or more lotion,cream, patch, or the like in accordance with any of the foregoing,wherein each patch contains a single unit dose for administration to asubject.

Imaging components can optionally be included and the packaging also caninclude written or web-accessible instructions for using theformulation. A container can include, for example, a vial, bottle,patch, syringe, pre-filled syringe, tube or any of a variety of formatswell known in the art for multi-dispenser packaging.

Administration and Dosing

The formulations provided herein can be topically administered in anyform. For administration for the treatment of skin conditions asufficient amount of the topical composition can be applied onto adesired area and surrounding skin, for example, in an amount sufficientto cover a desired area plus a margin of healthy skin or tissuesurrounding the desired area, if possible, for example, a margin ofabout 0.5 inches. A desired area can be an area of the skin affected byskin disorder in some embodiments. However, in other embodiments adesired area of the skin may be unaffected or healthy, e.g. skin nothaving any disorder or condition. Also, the formulations can be appliedto any skin surface, including for example, facial skin, and the skin ofthe hands, neck, chest and/or scalp.

For administration for the treatment of conditions that are not skindisorders, such as cancer and related disorders, gout, bladder andkidney stones and the like, there is usually no particular afflictedarea of the patient's skin and it is often desirable to administer theformulations provided herein to several skin surfaces of the patient ora large surface area to increase the amount of lotion applied.

In applying the formulations of the invention, the formulation itself issimply placed on the skin and spread across the surface and/or massagedto aid in penetration. The amount of formulation used is typicallysufficient to cover a desired surface area. In some embodiments, aprotective cover is placed over the formulation once it is applied andleft in place for a suitable amount of time, i.e., 5 minutes, 10minutes, 20 minutes or more; in some embodiments an hour or two. Theprotective cover can simply be a bandage including a bandage suppliedwith a cover that is impermeable to moisture. This essentially locks inthe contact of the formulation to the skin and prevents distortion ofthe formulation by evaporation in some cases. The composition may beapplied to the skin using standard procedures for application such as abrush, a syringe, a gauze pad, a dropper, or any convenient applicator.More complex application methods, including the use of delivery devices,may also be used, but are not required. In an alternative toadministering topically to intact skin, the surface of the skin may alsobe disrupted mechanically by the use of spring systems, laser poweredsystems, systems propelled by Lorentz force or by gas or shock wavesincluding ultrasound and may employ microdermabrasion such as by the useof sandpaper or its equivalent or using microneedles or electroporationdevices. Simple solutions of the agent(s) as well as the above-listedformulations that penetrate intact skin may be applied using occlusivepatches, such as those in the form micro-patches. External reservoirs ofthe formulations for extended administration may also be employed.

In an alternative to administering topically to intact skin, the surfaceof the skin may also be disrupted mechanically by the use of springsystems, laser powered systems, use of iontophoresis, systems propelledby Lorentz force or by gas or shock waves including ultrasound and mayemploy microdermabrasion such as by the use of sandpaper or itsequivalent or using microneedles or electroporation devices. Simplesolutions of the agent(s) as well as the above-listed formulations thatpenetrate intact skin may be applied using occlusive patches, such asthose in the form micro-patches. External reservoirs of the formulationsfor extended administration may also be employed.

Accordingly, in certain embodiments alternative methods of administeringone or more buffering agent, therapeutic compounds, agents, drugsthrough intact skin are provided. As nonlimiting examples, thesealternative methods might be selected from the following lists: on basisof working mechanism, spring systems, laser powered, energy-propelled,Lorentz force, gas/air propelled, shock wave (including ultrasound), onbasis of type of load, liquid, powder, projectile, on basis of drugdelivery mechanism, nano-patches, sandpaper (microdermabrasion),iontophoresis enabled, microneedles, on basis of site of delivery,intradermal, intramuscular, and subcutaneous injection. Other suitabledelivery mechanisms include, without limitation, microneedle drugdelivery, such as 3M Systems, Glide SDI (pushes drug as opposed to“firing” drug), MIT low pressure injectors, micropatches (single useparticle insertion device), microelectro mechanical systems (MEMS),dermoelectroporation devices (DEP), transderm ionto system (DEP), TTStransdermal therapeutic systems, membrane-moderated systems (drugreservoir totally encapsulated in a shallow compartment), adhesivediffusion-controlled system (drug reservoir in a compartment fabricatedfrom drug-impermable metallic plastic backing), matrix dispersion typesystem (drug reservoir formed by homogeneously dispersing drug solids ina hydrophilic or lipophilic polymer matrix molder into medicated disc),and microreservoir system (combination of reservoir and matrixdispersion-type drug delivery system).

It has been found, generally, that the requirements for effectivepenetration of the skin in the case of buffers as active agents are lessrestrictive than those required for alternative agents useful inpreventing cancer metastasis. In addition, although for theseindications delivery to the locus of the solid tumor, includingmelanoma, or melasma or gout is desirable, effective systemic pHalteration can be used as a way to diagnose the effectiveness ofpenetration when topical administration is employed.

The application method is determined by the nature of the treatment butmay be less critical than the nature of the formulation itself. If theapplication is to a skin area, it may be helpful in some instances toprepare the skin by cleansing or exfoliation. In some instances, it ishelpful to adjust the pH of the skin area prior to application of theformulation itself. The application of the formulation may be by simplemassaging onto the skin or by use of devices such as syringes or pumps.Patches could also be used. In some cases, it is helpful to cover thearea of application to prevent evaporation or loss of the formulation.

Where the application area is essentially skin, it is helpful toseal-off the area of application subsequent to supplying the formulationand allowing the penetration to occur so as to restore the skin barrier.A convenient way to do this is to apply a composition comprisinglinoleic acid which effectively closes the entrance pathways that wereprovided by the penetrants of the invention. This application, too, isdone by straightforward smearing onto the skin area or can be appliedmore precisely in measured amounts.

In some embodiments, the disclosure is directed to administering a localanesthetic to a subject transdermally and a formulation which containsan effective amount of anesthetic along with 25%-70% w/w or 30%-60% w/wor 30%-40% w/w of lecithin organogel typically wherein the lecithinorganogel comprises soy lecithin in combination with isopropyl palmitateor isopropyl myristate and benzyl alcohol in the range of 0.5%-20% w/wor 0.9%-2% w/w benzyl alcohol optionally including 1%-5% w/w or 2%-4%w/w menthol wherein the composition is topped off with a polar solution,typically an aqueous solution comprising 15%-50% w/w or 20%-40% w/w or20%-30% w/w poloxamer, typically Pluronic® or alternatively may be ananhydrous composition comprising bile salts such as deoxycholic acid orsodium deoxycholate in the range of 4%-8% w/w, typically 6% w/w and theremainder of the composition powdered nonionic detergent, typicallyPluronic®. The pH of the compositions is adjusted to 9-11, typically10-11. The formulations are applied to the desired area of the skin andmay be covered, for example, with Saran™ wrap for a suitable amount oftime. Following the treatment, the skin can be repaired by applying acomposition comprising linoleic acid.

A wide variety of therapeutic agents may be used in the formulations,including anesthetics, fat removal compounds, nutrients, nonsteroidalanti-inflammatory drugs (NSAIDs) agents for the treatment of migraine,hair growth modulators, antifungal agents, anti-viral agents, vaccinecomponents, tissue volume enhancing compounds, anti-cellulitetherapeutics, wound healing compounds, compounds useful to effectsmoking cessation, agents for prevention of collagen shrinkage, wrinklerelief compounds such as Botox®, skin-lightening compounds, compoundsfor relief of bruising, cannabinoids including cannabidiols for thetreatment of epilepsy, compounds for adipolysis, compounds for thetreatment of hyperhidrosis, acne therapeutics, pigments for skincoloration for medical or cosmetic tattooing, sunscreen compounds,hormones, insulin, corn/callous removers, wart removers, and generallyany therapeutic or prophylactic agent for which transdermal delivery isdesired. As noted above, the delivery may simply affect transport acrossthe skin into a localized subdermal location, such as treatment of nailfungus or modulation of hair growth or may effect systemic delivery suchas is desirable in some instances where vaccines are used.

In addition to the compositions and formulations of the invention perse, the methods may employ a subsequent treatment with linoleic acid. Astransdermal treatments generally open up the skin barrier, which is,indeed, their purpose, it is useful to seal the area of applicationafter the treatment is finished. Thus, treatment with the formulationmay be followed by treating the skin area with a composition comprisinglinoleic acid to seal off the area of application. The application oflinoleic acid is applicable to any transdermal procedure that results inimpairing the ability of the skin to act as a protective layer. Indeed,most transdermal treatments have this effect as their function is toallow carbonates to pass through the epidermis to the dermis at least,and, if systemic administration is achieved, through the dermis itself.

For administration of anesthetics as the therapeutic agent, the localanesthetic may be one or more of the following: benzocaine, lidocaine,tetracaine, bupivacaine, cocaine, etidocaine, mepivacaine, pramoxine,prilocaine, procaine, chloroprocaine, oxyprocaine, proparacaine,ropivacaine, dyclonine, dibucaine, propoxycaine, chloroxylenol,cinchocaine, dexivacaine, diamocaine, hexylcaine, levobupivacaine,propoxycaine, pyrrocaine, risocaine, rodocaine, and pharmaceuticallyacceptable derivatives and bioisosteres thereof. Combinations ofanesthetic agents may also be used. The anesthetic agent{s) are includedin the composition in effective amount(s). Depending on theanesthetic(s) the amounts of anesthetic or combination is typically inthe range of 1 w/w to 50% w/w. The compositions of the invention providerapid, penetrating relief that is long lasting. The pain to be treatedcan be either traumatic pain and/or inflammatory pain.

In one embodiment, the anesthetic is administered to relieve the painassociated with invasive fat deposit removal. Specific removal of fatdeposits has been attractive for both health and cosmetic reasons. Amongthe methods employed are liposuction and injection of a cytolytic agentfor fat such as deoxycholic acid (DCA). For example, a series of patentsissued or licensed to Kythera Biopharmaceuticals is directed to methodsand compositions for non-surgical removal of localized fat that involvesinjecting compositions containing DCA or a salt thereof. Representativeissued patents are directed to formulation (U.S. Pat. No. 8,367,649);method-of-use (U.S. Pat. Nos. 8,846,066; 7,622,130; 7,754,230;8,298,556); and synthetic DCA (U.S. Pat. No. 7,902,387).

In this aspect of the invention, conventional invasive fat removaltechniques are employed along with administering a pain-relievingeffective agent—typically lidocaine or related anesthetics viatransdermal administration. In some embodiments, the pain-relievingtransdermal formulation is applied to the area experiencing painimmediately before, during or immediately after the invasive fat-removalprocedure.

Additional therapeutic agents may be included in the compositions. Forexample, hydrocortisone or hydrocortisone acetate may be included in anamount ranging from 0.25% w/w to about 0.5% w/w. Menthol, phenol, andterpenoids, e.g., camphor, can be incorporated for cooling pain relief.For example, menthol may be included in an amount ranging from about0.1% w/w to about 1.0% w/w.

The compositions containing anesthetics are useful for temporary reliefof pain and itching associated with minor burns, cuts, scrapes, skinirritations, inflammation and rashes due to soaps, detergents orcosmetics, or, as noted above, pain associated with removal of fatdeposits.

The benefits of alkaline pH include higher penetration capability andadjustment of the active form of the fat dissolving compound when theanesthetic is used in conjugation therewith. For example, the pKa of thedeoxycholic acid is 6.58 and the pH of fat is neutral. When deoxycholicacid (DCA) is injected without buffering, it is approximately anequilibrium between the protonated and unprotonated forms. Utilizingformulations with high pH buffering shifts the balance significantly tounprotonated form making the DCA more water soluble and more likely toemulsify fats.

The formulations can be applied in a single, one-time application, oncea week, once a bi-week, once a month, or from one to twelve times daily,for a period of time sufficient to alleviate a condition, disease,disorder, symptoms, for example, for a period of time of one week, from1 to 12 weeks or more, from 1 to 6 weeks, from 2 to 12 weeks, from 2 to12 weeks, from 2 to 8 weeks, from 2 to 6 weeks, from 2 to 4 weeks, from4 to 12 weeks, from 4 to 8 weeks, or from 4 to 6 weeks. The presentcompositions can be administered, for example, at a frequency of onceper day to hourly if needed. The presently described formulations can betopically administered once or more per day for a period of time from 1week to 4 weeks, of from 1 week to 2 weeks, for 1 week, for 2 weeks, for3 weeks, for 4 weeks, or for 4 weeks or more. In some instances, it mayalso be desirable to continue treatment indefinitely for example toinhibit or prevent carcinogenesis or for improving, extending theduration of remission, or maintaining remission of a cancer or anotherdisease or disorder. A suitable administration for a formulationcomprising a skin cream, lotion or ointment, for example is once, twice,three, four times daily, or hourly if needed.

The formulations provided herein can be applied in a therapeuticallyeffective amount. Suitable amounts, for example, per application caninclude, for example, from about 1 gram to about 500_([SB1]) grams; fromabout 1 gram to about 10 grams; from about 10 grams to about 25 grams;from about 10 grams to about 50 grams; from about 10 grams to about 100grams; from about 10 grams to about 200 grams; from about 10 grams toabout 350 grams; from about 10 grams to about 500 grams; from about 20grams to about 500 grams; from about 20 grams to about 350 grams; fromabout 20 grams to about 200 grams; from about 20 grams to about 100grams; from about 20 grams to about 90 grams; from about 20 grams toabout 80 grams; from about 20 grams to about 70 grams; from about 20grams to about 60 grams; from about 20 grams to about 50 grams; fromabout 30 grams to about 100 grams; from about 30 grams to about 80grams; from about 30 grams to about 70 grams; or from about 30 grams toabout 60 grams. Alternatively, suitable amounts, for example, perapplication can include, for example, at least 5 grams; at least 10grams; at least 15 grams; at least 20 grams; at least 25 grams; at least30 grams; at least 35 grams; at least 40 grams; at least 50 grams; atleast 55 grams; at least 60 grams; at least 65 grams; at least 70 grams;at least 75 grams; at least 80 grams; at least 85 grams; at least 90grams; at least 100 grams; or more.

If desired, other therapeutic agents can be employed in conjunction withthose provided in the above-described compositions. The amount of activeingredients that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host treated, the natureof the disease, disorder, or condition, and the nature of the activeingredients.

It is understood that a specific dose level for any particular patientwill vary depending upon a variety of factors, including the activity ofthe specific active agent; the age, body weight, general health, sex anddiet of the patient; the time of administration; the rate of excretion;possible drug combinations; the severity of the particular conditionbeing treated; the area to be treated and the form of administration.One of ordinary skill in the art would appreciate the variability ofsuch factors and would be able to establish specific dose levels usingno more than routine experimentation.

Pharmacokinetic parameters such as bioavailability, absorption rateconstant, apparent volume of distribution, unbound fraction, totalclearance, fraction excreted unchanged, first-pass metabolism,elimination rate constant, half-life, and mean residence time can bedetermined by methods well known in the art.

A formulation in accordance with the subject matter described herein maybe a topical dosage form packaged in, for example, a multi-use orsingle-use package, including for example, a tube, a tottle, a pump, acontainer or bottle, a vial, a jar, a packet, or a blister package.

Single dosage kits and packages containing a once per day amount of thetopical formulation may be prepared. Single dose, unit dose, andonce-daily disposable containers of the topical formulation are alsoprovided.

The present topical formulation remains stable in storage for periodsincluding up to about 5 years, between about 3 months and about 5 years,between about 3 months and about 4 years, between about 3 months andabout 3 years, and alternately any time period between about 6 monthsand about 3 years.

A topical formulation described herein remains stable for up to at least3 years at a temperature of less than or equal to 40° C. In anembodiment, the presently described topical formulation remains stablefor at least 2 years at a temperature of less than or equal to 40° C. Inan embodiment, the presently described formulation or emulsion remainsstable for at least 3 years at a temperature of less than or equal to40° C. and at a humidity of up to 75% RH, for at least 2 years at atemperature of less than or equal to 40° C. and at a humidity of up to75% RH, or for at least 3 years at a temperature of less than or equalto 30° C. and at a humidity of up to 75% RH. In a further embodiment,the presently described biocompatible composition in accordance with thesubject matter described herein remains stable for an extended period oftime when packaged in a multi-use container such as a bottle dispenseror the like, and exhibits equal to or even greater stability whenpackaged in a single-use package.

In another aspect, the pharmaceutical composition of certain embodimentscomprises a daily dose of a pH modulating composition or buffer (e.g.sodium bicarbonate as a topical formulation). A daily dose for topicalor transdermal administration of any given pH modulating compounddepends on the compound and animal and may be easily determined by theskilled artisan, a suitable amount is about 1 mg/kg to about 5 g/kg, andmore typically the daily dose is about 10 mg/kg to about 5 g/kg, about25 mg/kg to about 2000 mg/kg, about 50 mg/kg to about 2000 mg/kg, about25 mg/kg to about 1000 mg/kg, about 50 mg/kg to about 1000 mg/kg, about100 mg/kg to about 700 mg/kg, about 100 mg/kg to about 500 mg/kg, about150 mg/kg to about 500 mg/kg, about 150 mg/kg to about 400 mg/kg, about200 mg/kg to about 500 mg/kg, about 200 mg/kg to about 450 mg/kg, about200 mg/kg to about 400 mg/kg, about 250 mg/kg to about 450 mg/kg, about250 mg/kg to about 400 mg/kg, about 250 mg/kg to about 350 mg/kg, andabout 275 mg/kg to about 325 mg/kg.

Alternatively, a suitable daily dose for topical or transdermaladministration of a pH modulating composition or buffer (e.g. sodiumbicarbonate) is at least about 1 mg/kg, at least about 10 mg/kg, atleast about 25 mg/kg, at least about 30 mg/kg, at least about 35 mg/kg,at least about 40 mg/kg, at least about 41 mg/kg, at least about 42mg/kg, at least about 43 mg/kg, at least about 44 mg/kg, at least about45 mg/kg, at least about 46 mg/kg, at least about 47 mg/kg, at leastabout 48 mg/kg, at least about 49 mg/kg, at least about 50 mg/kg, atleast about 55 mg/kg, at least about 60 mg/kg, at least about 65 mg/kg,at least about 70 mg/kg, at least about 75 mg/kg, at least about 80mg/kg, at least about 90 mg/kg, at least about 100 mg/kg, at least about125 mg/kg, at least about 150 mg/kg, at least about 160 mg/kg, at leastabout 170 mg/kg, at least about 175 mg/kg, at least about 180 mg/kg, atleast about 190 mg/kg, at least about 200 mg/kg, at least about 225mg/kg, at least about 250 mg/kg, at least about 275 mg/kg, at leastabout 300 mg/kg, at least about 325 mg/kg, at least about 350 mg/kg, atleast about 375 mg/kg, at least about 400 mg/kg, at least about 425mg/kg, at least about 450 mg/kg, at least about 475 mg/kg, at leastabout 500 mg/kg, at least about 550 mg/kg, at least about 600 mg/kg, atleast about 700 mg/kg, at least about 800 mg/kg, at least about 900mg/kg, at least about 1 g/kg, at least about 2 g/kg, at least about 3g/kg, or at least about 5 g/kg.

Alternatively, a suitable dose for topical or transdermal administrationof a pH modulating formulation or buffer (e.g. sodium bicarbonate) forsubject (e.g. a human patient) is at least about 100 mg, at least about500 mg, at least about 1 g, at least about 5 g, at least about 10 g, atleast about 15 g, at least about 16 g, at least about 17 g, at leastabout 18 g, at least about 19 g, at least about 20 g, at least about 21g, at least about 22 g, at least about 23 g, at least about 24 g, atleast about 25 g, at least about 26 g, at least about 27 g, at leastabout 28 g, at least about 29 g, at least about 30 g, at least about 35g, at least about 40 g, at least about 45 g, at least about 50 g, atleast about 60 g, at least about 75 g, at least about 100 g, at leastabout 200 g, at least about 500 g, or at least about 1.0 kg. This doesmay be administered daily, twice a day, three times a day, four times aday, five times a day, or more than five times a day.

In another aspect, in certain embodiments a pH modulating composition orbuffer (e.g. sodium bicarbonate) is administered topically ortransdermally such that the dose results in a subject intake of at leastabout 0.1 nmol/hr/Kg, at least about 0.5 nmol/hr/Kg, at least about 0.7nmol/hr/Kg, at least about 1.0 nmol/hr/Kg, at least about 1.1nmol/hr/Kg, at least about 1.2 nmol/hr/Kg, at least about 1.3nmol/hr/Kg, at least about 1.4 nmol/hr/Kg, at least about 1.5nmol/hr/Kg, at least about 1.6 nmol/hr/Kg, at least about 1.7nmol/hr/Kg, at least about 1.8 nmol/hr/Kg, at least about 1.9nmol/hr/Kg, at least about 2.0 nmol/hr/Kg, at least about 2.5nmol/hr/Kg, at least about 3.0 nmol/hr/Kg, at least about 3.5nmol/hr/Kg, at least about 4.0 nmol/hr/Kg, at least about 5 nmol/hr/Kg,at least about 10 nmol/hr/Kg, at least about 25 nmol/hr/Kg, at leastabout 50 nmol/hr/Kg, at least about 100 nmol/hr/Kg, at least about 500nmol/hr/Kg, or at least about 1 μmol/hr/Kg,

In another aspect, in certain embodiments a pH modulating composition orbuffer (e.g. sodium bicarbonate) is administered topically ortransdermally such that the dose results in a peak plasma concentrationof a buffering or pH modulating compound ranges from about 1 μg/ml to 50μg/ml, about 5 μg/ml to about 45 μg/ml, about 5 μg/ml to about 40 μg/ml,about 5 μg/ml to about 35 μg/ml, about 5 μg/ml to about 30 μg/ml, about5 μg/ml to about 25 μg/ml, about 1 μg/ml to about 45 μg/ml, about 1μg/ml to about 40 μg/ml, about 1 μg/ml to about 35 μg/ml, about 1 μg/mlto about 30 μg/ml, about 1 μg/ml to about 25 μg/ml, about 1 μg/ml toabout 20 μg/ml, about 1 μg/ml to about 15 μg/ml, about 1 μg/ml to about10 μg/ml, about 1 μg/ml to about 9 μg/ml, about 1 μg/ml to about 8μg/ml, about 1 μg/ml to about 7 μg/ml, about 1 μg/ml to about 6 μg/ml,and about 1 μg/ml to about 5 μg/ml.

In another aspect, in certain embodiments a pH modulating composition orbuffer (e.g. sodium bicarbonate) is administered topically ortransdermally so that plasma concentration ranges from about 1 ng/ml to500 μg/ml, about 10 ng/ml to 500 μg/ml, about 100 ng/ml to 500 μg/ml,about 1 μg/ml to 500 μg/ml, about 10 μg/ml to 500 μg/ml, about 25 μg/mlto 500 μg/ml, about 25 μg/ml to about 450 μg/ml, about 25 μg/ml to about400 μg/ml, about 25 μg/ml to about 350 μg/ml, about 25 μg/ml to about300 μg/ml, about 25 μg/ml to about 250 μg/ml, about 50 μg/ml to about500 μg/ml, about 55 μg/ml to about 500 μg/ml, about 60 μg/ml to about500 μg/ml, about 65 μg/ml to about 500 μg/ml, about 70 μg/ml to about500 μg/ml, about 75 μg/ml to about 500 μg/ml, about 80 μg/ml to about500 μg/ml, about 85 μg/ml to about 500 μg/ml, about 90 μg/ml to about500 μg/ml, about 95 μg/ml to about 500 μg/ml, about 100 μg/ml to about500 μg/ml, about 110 μg/ml to about 500 μg/ml, about 120 μg/ml to about500 μg/ml, about 130 μg/ml to about 500 μg/ml, about 140 μg/ml to about500 μg/ml about 150 μg/ml to about 500 μg/ml, about 160 μg/ml to about500 μg/ml, about 170 μg/ml to about 500 μg/ml, about 180 μg/ml to about500 μg/ml, about 200 μg/ml to about 500 μg/ml, about 200 μg/ml to about490 μg/ml, about 200 μg/ml to about 480 μg/ml, about 200 μg/ml to about470 μg/ml, about 200 μg/ml to about 460 μg/ml, about 200 μg/ml to about450 μg/ml, about 200 μg/ml to about 440 μg/ml, about 200 μg/ml to about430 μg/ml, or about 200 μg/ml to about 400 μg/ml.

In further embodiments, a pH modulating composition or buffer (e.g.sodium bicarbonate) is administered topically or transdermally so thatplasma concentration is at least 10 ng/ml, at least 25 ng/ml, at least50 ng/ml, at least 100 ng/ml, at least 250 ng/ml, at least 0.5 μg/ml, atleast 0.75 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 3 μg/ml,at least 4 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 7 μg/ml,at least 8 μg/ml, at least 9 μg/ml, at least 10 μg/ml, at least 15μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 30 μg/ml, at least35 μg/ml, at least 40 μg/ml, at least 45 μg/ml, at least 50 μg/ml, atleast 55 μg/ml, at least 60 μg/ml, at least 65 μg/ml, at least 70 μg/ml,at least 75 μg/ml, at least 80 μg/ml, at least 85 μg/ml, at least 90μg/ml, at least 95 μg/ml, at least 100 μg/ml or more than 100 μg/ml.

In another aspect, a pH modulating compound or buffer (e.g. sodiumbicarbonate) is administered topically or transdermally so that peakplasma concentration is reached in 10 min, 15 min, 20 min, 30 min, 45min, 60 min, 75 min, 90 min, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr,10 hr, 12 hr or 24 hr after administration.

Aspects of the present specification disclose that the symptomsassociated with a disease or disorder described herein are reduced by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% and the severity associatedwith a disease or disorder described herein is reduced by at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%. Aspects of the present specificationdisclose the symptoms associated with disease or disorder are reduced byabout 10% to about 100%, about 20% to about 100%, about 30% to about100%, about 40% to about 100%, about 50% to about 100%, about 60% toabout 100%, about 70% to about 100%, about 80% to about 100%, about 10%to about 90%, about 20% to about 90%, about 30% to about 90%, about 40%to about 90%, about 50% to about 90%, about 60% to about 90%, about 70%to about 90%, about 10% to about 80%, about 20% to about 80%, about 30%to about 80%, about 40% to about 80%, about 50% to about 80%, or about60% to about 80%, about 10% to about 70%, about 20% to about 70%, about30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

The formulations as described herein can be used in the manufacture ofmedicaments and for the treatment of humans and other animals byadministration in accordance with conventional procedures.

EXPERIMENTAL EXAMPLES

The compositions and methods described herein will be further understoodby reference to the following examples, which are intended to be purelyexemplary. The compositions and methods described herein are not limitedin scope by the exemplified embodiments, which are intended asillustrations of single aspects only. Any methods that are functionallyequivalent are within the scope of the invention. Various modificationsof the compositions and methods described herein in addition to thoseexpressly described herein will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications fall within the scope of the invention.

The following examples are intended to illustrate but not to limit theinvention.

Example 1—Specific Formulations for Administration of Buffering Agents

The following compositions have been prepared and are found useful inthe methods of the invention. In the tables below, “LIP” representslecithin organogel comprised of a 1:1 molar mixture of soy lecithincontaining 96% phosphatidyl choline and isopropyl palmitate; “BA”represents benzyl alcohol; PLU-F127 represents the detergent poloxamerF127 granules; PLU-Water represents PLU-F127 dissolved in deionizedwater. (Alternatively, commercially available Pluronic F127 30% gelcould be used) and Durasoft® is a commercially available form ofemulsifier.

In the tables below, sodium bicarbonate and sodium carbonate weresupplied as such. Tris buffer at pH 8.1 was used. The phosphate “buffer”was supplied as Na H₂ PO₄. The percentages are wt/wt i.e., weightpercentages.

TABLE 1 Bicarbonate Formulations IB IIB LIP 30.00% 25.00% Ethanol  1.50% 0.00% BA  1.00%  1.00% Menthol  0.50%  0.90% NaBicarbonate 33.50% 5.76% PLU-F127 10.05% 10.50% Water in gel with PLU 23.45% 24.50%Additional Water 30.34% Durasoft  2.00% TOTAL 100.00%  100.00% 

TABLE 2 Carbonate Formulations IC IIC IIIC IVC LIP 25.00% 25.00% 30.00%40.00% Ethanol  0.00%  0.00%  1.50%  1.50% BA  1.00%  1.00%  1.00% 1.00% Menthol  0.90%  0.90%  0.50%  0.50% Sodium Carbonate  9.36%16.10% 33.50% 11.00% PLU-F127 10.50% 10.00% 10.05% 13.50% Water in gelwith PLU 24.50% 23.33% 23.45% 31.53% Additional Water 26.74% 22.67% 0.00%  0.00% Durasoft  2.00%  1.00%  0.00%  1.00% TOTAL 100.00% 100.00%  100.00%  100.00% 

TABLE 3 Tris Formulations IT IIT IIIT IVT LIP 25.00% 25.00% 30.00% 38.40Ethanol  0.00%  0.00%  1.50% 1.50 BA  1.00%  1.00%  1.00% 1.00 Menthol 0.90%  0.90%  0.50% 0.50 TRIS buffer, pH 8.1 13.37% 23.00% 33.50% 14.93PLU-F127 10.50% 10.00% 10.05% 12.80 Water in gel with PLU 24.50% 23.33%23.45% 29.87 Additional Water 22.73% 15.77%  0.00% 0.00 Durasoft  2.00% 1.00%  0.00% 1.00 TOTAL 100.00%  100.00%  100.00%  100.00

TABLE 4 Phosphate Formulations IP IIP IIIP IVP LIP 25.00% 25.00% 30.00%37.25 Ethanol  0.00%  0.00%  1.50% 1.50 BA  1.00%  1.00%  1.00% 1.00Menthol  0.90%  0.90%  0.50% 0.50 Monosodium phosphate 16.02% 27.55%33.50% 17.36 PLU-F127 10.50% 10.00% 10.05% 12.42 Water in gel with PUT24.50% 23.33% 23.45% 28.97 Additional Water 20.08% 11.22%  0.00% 0.00Durasoft  2.00%  1.00%  0.00% 1.00 TOTAL 100.00%  100.00%  100.00% 100.00

TABLE 5 Carbonate, TRIS, Phosphate Combination Formulations Phos—lowPhos—high III IV LIP 25.00% 25.00% 30.00% 37.25 Ethanol  0.00%  0.00% 1.50% 1.50 BA  1.00%  1.00%  1.00% 1.00 Menthol  0.90%  0.90%  0.50%0.50 Sodium Carbonate  5.30%  5.30%  5.30% 5.30 TRIS (121.14)  6.00% 6.00%  6.00% 6.00 Monosodium phosphate  6.00%  6.00%  6.00% 6.00PLU-F127 10.50% 10.00% 10.05% 12.42 Water in gel with PLU 24.50% 23.33%23.45% 28.97 Additional Water 12.14% 21.47% 16.20% 0.06 DURASOFT  2.00% 1.00%  0.00% 1.00 TOTAL 100.00%  100.00%  100.00%  100.00

TABLE 6 Alternative Buffers 25 28 29 A(2) B(2) C(2) LIP  6.00% 12.00%12.00% 14.00% 15.00% 18.00% BA  1.00%  1.00%  1.00%  1.00%  1.00%  1.00%Menthol  0.50%  0.50%  0.50%  0.25%  0.25%  0.50% Durasoft  1.50%  1.50% 1.00% Pluronic Granules  4.20%  4.20%  4.20%  5.40%  2.10%  3.60% Water37.80% 37.80% 40.30% 31.60% 29.65% 41.90% Sodium Carbonate  7.00%  7.00% 7.00% — —  3.00% Sodium Bicarbonate 28.00% 28.00% 28.00% 32.50% 32.50%15.00% propylene glycol  3.00%  3.00%  6.00% 10.00%  5.00% almond oil 3.00%  3.00%  3.00%  4.00%  3.00%  4.50% zinc oxide  0.25%  0.50% cetylalcohol  2.00%  2.00%  2.00%  2.00%  3.00%  3.00% lecithin  3.00% cetiolultimate (mixture of tridecane and undecane) ethanol  1.50%  1.50% 1.50%  1.50%  1.50%  1.50% EGTA  0.50%  1.00% Sodium Decanoate  1.00%TOTAL 100.00%  100.00% 100.00%  100.00%  100.00%  100.00% 

TABLE 7 Anhydrous Formulations 26 27 30 menthol  0.20%  0.20%  0.50%Ethanol  2.00%  2.00%  1.50% Benzyl Alcohol  8.00%  8.00%  1.00% CetylAlcohol  2.00% Almond Oil  5.00%  5.00%  3.00% Lecithin 15.90% 15.90% 6.00% Lipmax 15.90% 15.90%  6.00% Propylene Glycol  3.00%  3.00%  0.00%F127 Pluronic Powder 16.00% 16.00%  4.20% Water 39.80% SodiumBicarbonate 26.00%  0.00% 28.00% Sodium Carbonate  7.00% 33.00%  7.00%Durasoft  1.00%  1.00%  1.00% 100.00%  100.00%  100.00% 

Example 2—Topical Administration of Bicarbonate

24 NCR nude 5 week old male mice were used in this study, divided intofour groups of six mice each. Topical compositions were applied to theback of each mouse from hip to shoulder three times per day for 8successive days for a total of 24 applications. A control group wasadministered sodium bicarbonate in water by mouth. The groups are asfollows:

Group 1. Sodium bicarbonate in H₂0 Group 2. transdermal bicarbonate Dose#1 (30 μL)(10 μL × 3 doses) Group 3. transdermal bicarbonate Dose #2(220 μL)(73 μL × 3 doses) Group 4. transdermal bicarbonate Dose #3 (1110μL)(2 × 185 μL × 3 doses)

The transdermal formulations comprise penetrants to result in theformulations as follows: LIP-30.0%, EtOH-1.5%, BA-1.0%, menthol-0.5%,sodium bicarbonate-33.5%, PLU-F127-10.1%, PLU-water-23.5%; i.e.,Formulation IB in Table 1. The concentration of sodium bicarbonate inthe control group was 200 mM and the consumption was ad libidum. Theconcentration of sodium bicarbonate in the transdermal formulations was33.5% wt/wt.

Urine samples were collected at one hour, three hours and six hoursafter the first drug application and stored at 4° C. for subsequent pHdetermination. On days 2-12 urine was collected twice daily—prior to thefirst application and 15 minutes after the last application. The micewere sacrificed one hour after the last drug application on day 8 andthe back skin was harvested and placed on bibulous paper.

To set a base line for pH without dosing, prior to beginning theprotocol, urinary pH was measured at three time points during a singleday in seven mice at 0900, five mice at 1300 and four mice at 1630. ThepH of the urine had an overall mean value of 5.57 which did not varyover this time period.

As shown in FIG. 1, all of the groups showed an increase in urine pHover the first six hours of treatment. The most significant increaseoccurred in Group 3 which was administered 220 microliters of theformulation.

Although the study was designed to be conducted for two weeks (Mon-Fri),it was terminated after day eight because the members of Group 2 (1,110microliter) developed skin irritation; this was shown even in the lowdose group receiving 30 microliters, i.e., Group 2.

FIG. 2 shows the urine values of pH over the course of the eight daystudy. Although there was some variation, the group receiving thehighest dosage (Group 4) was able to maintain a high pH over the courseof the study.

The study was repeated using the formulations 25, 28 and 29 in Table 6with the results shown in Table 8 and using the formulations in Table 7with results shown in

FIG. 3.

TABLE 8 Mean urine pH at two collection time points on day one andoverall. urine 1 urine 2 overall SB water 6.30 6.03 6.15 25 6.36 6.776.62 28 6.92 6.86 6.89 29 6.82 7.31 6.97

As shown, transdermal administration was more effective than oraladministration.

Example 3—Transdermal Absorption in Humans

Healthy human Subjects Aged 18-60 were enrolled in a double-blinded,placebo controlled, randomized and cross-over in designed study. Thesubjects applied 0.6 g/kg of body weight of formulation of Table 9, perrandomization group, as follows: legs from ankle to top of thighs; armsfrom wrist to shoulder (including the deltoids) or drank 0.13 ounces ofwater per kg bodyweight (equates to about 8 ounces for a 140 poundsubject) at 15 min, 1 hr 15 min, 2 hours and 15 min, 3 hours and 15 minsto control for dilution of urine.

TABLE 9 LIP 30.0% Ethanol  1.5% BA  1.0% Menthol (or limonene for  0.5%lipophilic) NaBicarbonate 33.5% PLU-F127 10.1% PLU-Water 23.5%

At hourly intervals (at 1, 2, 3, and 4 hours) from start time ofapplication, subjects collected 10-20 ml (approximately a Tablespoon) ofurine and the pH was determined. The results are shown in Table 10.

TABLE 10 Average Urine pH values for each treatment and time pointBaseline 1 hour 2 hours 3 hours 4 hours Product (n = 20) (n = 20) (n =20) (n = 19) (n = 20) Control (oral) 5.75 5.95 6.01 6.08 6.01Formulation 5.86 611 6.27 6.24 6.23

No adverse effects were shown and the transdermal formulationout-performed oral administration.

Example 4—Treatment of Gout in Humans

A randomized, double-blinded, placebo controlled study of the efficacyand safety of a topical alkalinizing treatment for reducing painassociated with an acute gout flare was conducted to determine iftopical application of sodium bicarbonate and menthol in a transdermaldelivery system can effectively and safely reduce pain associated withan acute gout flare and if time to resolution is shortened.

40 subjects with a past history of gout and presenting in the clinicwith a gout attack that started within 36 hours and who had beenprescribed cochicine, aged 18+ were employed randomized into groups of20.

The formulations used are shown in Table 11.

TABLE 11 GoutFormulations Ingredient Control Active Menthol  0.00% 0.50% Ethanol  1.50%  1.50% Benzyl Alcohol  1.00%  1.00% Cetyl Alcohol 2.00%  2.00% Almond Oil  3.00%  3.00% Lecithin 10.00%  7.00% IPP 10.00% 7.00% Propylene Glycol  5.00%  5.00% Poloxamer/Pluronic Powder  9.00% 5.40% Water 57.50% 33.60% Sodium Bicarbonate  0.00% 33.00% DurosoftPK-SG  1.00%  1.00% 100.00%  100.00% 

Subjects applied 10 ml of Control or Active cream to the entire limb ofeach affected joint three times a day. One identified “target joint” andup to 2 additional joints were followed.

The subjects reported joint pain as measured using a 11-point scale(0-10, with 0 as “no pain” and 10 as “worst possible pain”). Thereduction in pain in the target joints for the Active group (twosubjects) was −3.0 points both after 30 minutes and 24 hours and −3.5points at 4 days and 6 days Subjects in the ControlGroup showed noreduction at 30 minutes and 2 days and only −1 point after 24 hours andat 4 and 6 days. The maximum reduction in pain in a secondary joint forthe Active group was −2 points, versus less or no reduction in pain forthe Control group.

The reduction in pain associated with Active product use was observed asearly as 15 minutes after product application.

A follow-up study was performed. This study was double-blinded,randomized, placebo controlled, and parallel group design. Fortysubjects, female and male, aged 18+, with a clinical diagnosis of gout,history of uric acid >6.8 mg/dl, on stable medication regimen,presenting in the clinic within 36 hours of initiation of acute goutattack and prescribed colchicine were included. The key exclusioncriteria for the study included >stage 3 kidney disease, tophaceousgout, and recent/concurrent initiation of other pain medications (e.g.NSAID, corticosterioids).

Subjects were randomized to receive placebo lotion or sodium bicarbonatetransdermal lotion (33% sodium bicarbonate and 0.5% menthol) andinstructed to apply to the entire limb of up to three affected joints (atarget joint and up to two other joints). Outcome measures included painusing a numeric rating scale (0-10), time to resolution (defined as 50%reduction in pain) range of motion and satisfaction with the treatment.Time-points were baseline, 15 & 30 min, and 1, 2, 4, 6, 8, 10, 12, and14 days.

The data was analyzed for 5 control and 4 active subjects. FIG. 4 showsthe % reduction in pain scores over time in the target joint. A similareffect was observed for other involved joints. Pain resolution was notreached by 3 control subjects, but was an average of 7 days in the twowho resolved. All subjects in the active group resolved, with theaverage being 3.25 days. Range of motion improvement at the lasttime-point collected was 50% for control and 100% for active. Subjectsatisfaction was 67% reporting ‘Neutral’ and 33% ‘Satisfied’ in controland 67% ‘Extremely Satisfied’ and 33% ‘Neutral’ for active. No treatmentrelated adverse events were reported. The data indicates that topicaltransdermal delivery of sodium bicarbonate in a lotion form as providedherein may safely and significantly reduce pain, as early as 15 min, andspeed resolution of acute gout attacks. FIG. 5 shows the target jointpain scores from zero to 8, measured starting at 15 min and over a 14day period.

Subjective pain scores of multiple joints that were identified as‘target joints’ were collected from patients in response to treatment tomeasure effectiveness. Target joints are identified by thecharacteristics of level of pain, pain experience similar to previousgout attacks, etc. Target joints included the left ankle, leftmetatarsophalangeal joint, left great toe, right knee, left 1st MTP,right 1st MTP, left 4^(th) MT joint, right wrist, left elbow, left handinterphalangeal joints, left knee, right foot, right wrist, right 4^(th)TMT, and left 1st MTP and pain of these joints was measured in responseto treatment of a patient.

FIG. 18—Gout joint pain measurements.

Pain scores (0-10 scale with 0 being no pain and 10 being the worstpossible pain) were collected from patients at the time points indicatedfollowing treatment of the entire limb that the target joint is locatedon with a transdermal buffer formulation with 0.5% menthol or a controlformulation (same base without the buffer or menthol).

FIG. 19—Gout joint pain measurements. FIG. 19 presents the same data asFIG. 18 and adds the % change from baseline for each timepoint.

Example 5—Treatment of Melasma in Humans

A randomized, double-blind study was conducted evaluating tranexamicacid in a transdermal delivery system, alone or with skin turnoveragents for improvement of melasma to evaluate the efficacy and safetyof 1) tranexamic acid (TA), 2) TA and 5% glycolic acid (GA) and 3) TAused in combination with retinoic acid (RA) to control for theimprovement of melasma. All the formulations including Control arealkaline. The formulations used are shown in Table 14.

TABLE 14 Melasma Formulations Active Active Ingredient Control (TA)(TA + GA) Tranexamic Acid  0.000%  6.000%  6.000% Glycolic Acid  0.000% 0.000%  5.000% Sodium Carbonate  1.060%  1.000%  1.000% Triethanolamine 0.000%  0.000%  1.000% Lecithin 13.300% 12.500% 12.500% IPP 13.300%12.500% 12.500% Benzyl Alcohol  1.060%  1.000%  1.000% Durosoft PK-SG 1.060%  1.000%  1.000% Poloxamer/Pluronic  9.255%  8.700%  6.900%Powder Water 60.745% 57.100% 52.900% Crisp Fruit Fragrance  0.220% 0.200%  0.200% 100.000%  100.000%  100.000% 

36 women aged 18-65 with moderate to severe melasma were divided intogroups of 12 each, and the subjects applied the formulations twice dailyat the site of the melasma. The Groups are as follows: Group 1: Control;Group 2: Tranexamic acid (TA) 6% Formulation; Group 3: TA 6%+GlycolicAcid 5% (GA) Combined Formulation; Group 4: TA 6% Formulation andRetinioic Acid 0.25% (RA) (separate products, with TA appliedimmediately before RA).

Outcome measures were by Standardized 2D photography, 5 views-frontal,45 and 90 degrees and included blinded Investigator Global Assessment ofImprovement compared to baseline; subject Self-Assessment of Improvementcompared to baseline and VISIA System Photography (one site). Adverseevents were evaluated as well.

Follow-ups were by a 3 day phone call and visits at 2, 4, 8 and 12weeks. In all these groups including Control had 50%-65% of subjectsshowed reduction in pigment after 30 days both by independent assessmentand self-assessment. The results were generally improved for Groups 3and 4 compared to Group 2—at 30 days independent assessment found 50% ofGroup 2 subjects improved, and 60% and 65% of subjects in Groups 3 and 4respectively. (Self-assessment results showed little differences all atabout 60%.)

Example 6—Administration of Bicarbonate Topically Using SelectedFormulations

An objective of this series of studies was to compare differenttransdermal compounds to determine which compound, dose, concentrationcombinations induce the highest urine with the lowest levels of skinirritation. Presented below are data and results from severalexperiments that determine and compare various formulations in terms ofeffectiveness for treatment and side effects, and in particulartolerance from skin irritation.

In the first study, twenty-four 6 week old female SCID mice were used inthis study, divided into four groups of six mice each. The backs of micewere treated with hair removal compound before topical formulations wereapplied. Topical formulations were then applied to the back of eachmouse from hip to shoulder three times per day at a dose of 50 μl,totaling 150 μl for 3 consecutive days. Urine samples were collectedtwice daily, one in the morning and one in the afternoon and stored at4° C. for subsequent pH determination. The transdermal formulations fromStudy 1 comprise formulations as follows: Group A, 15.8% sodiumbicarbonate in water; Group B, 29% lysine in water; Group C, 9.3% SodiumPhosphate in water; Group D, 23.4% Tris in water (see Table 15).Pre-dosing urine pH from 15 mice was 5.97±0.06 (mean±SEM). Thetransdermal formulations from cohort Groups A-D are shown in Table 15.

TABLE 15 Buffer Formulations Tested Cohorts Buffer formulation Totaldose/day Group A 15.8% sodium carbonate 150 μl Group B 29% lysine 150 μlGroup C 9.3% trisodium phosphate 150 μl Group D 23.4% TRIS 150 μl

In all cohort groups the formulations absorbed quickly and easily.Formula A caused severe skin irritation after the first day and scabbingby the end of the third day in the Group A cohort mice (not shown).However, formulation A is the only one tested in Table 15 thatsignificantly increased urine pH after the second day of dosing, andcyclic dinural variation was evident by day 2 (see FIG. 6).

In a second study, a variation of the above study was performed with theexclusion of Formula A in which the mice received larger doses to testthe effect of larger doses (100 μl/dose, 3 times a day). The totaldose/day in these cohort mice was 300 μl/day. Formula A was excludedfrom further testing because, while it was effective in increasing urinepH, it caused sever skin irritation and intolerance in mice. Thetransdermal formulations from Study 2 included the followingformulations: Group B, 29% lysine in water; Group C, 9.3% SodiumPhosphate in water; Group D, 23.4% Tris in water (see Table 16).

TABLE 16 Buffer Formulations Tested Cohorts Buffer formulation Totaldose/day Group B 29% lysine 300 μl Group C 9.3% trisodium phosphate 300μl Group D 23.4% TRIS 300 μl

Pre-dosing urine pH from 10 mice was 5.58±0.04 (mean±SEM). Thetransdermal agent absorbed quickly and easily in all cohort groups(A-D). No skin irritation observed in any of the cohorts. As illustratedin FIG. 7, the results show that compounds C and D increased urine pH atvarious intervals. Urine collected for the 2nd time on day 1 was highestin pH in mice treated with compound C (FIG. 7). Urine collected for the2nd time on day 2 was highest in pH in mice treated with compound D(FIG. 7).

In the third study, a variation of study II above was then performedwith a higher total dose/day to test the effect of larger doses (100μl/dose, 3 times a day). The total does/day in these cohort mice was 300μl/day. Formula A was excluded from further testing because, while itwas effective in increasing urine pH, it caused sever skin irritationand intolerance in mice. The transdermal formulations from Study 2comprised formulations as follows: Group B, 29% lysine in water; GroupC, 9.3% Sodium Phosphate in water; Group D, 23.4% Tris in water (seeTable 16). Pre-dosing urine from 10 mice was 5.58±0.04 (mean±SEM). Thetransdermal agent absorbed quickly and easily in all cohort groups(B,C,D). There was no skin irritation observed in any of the cohorts.The results are shown in FIG. 7, where it can be seen that compounds Cand D increased urine pH at various intervals. Urine was collected forthe 2nd time on day 1 was highest in pH in mice treated with compound C(FIG. 7). Urine collected for the 2nd time on day 2 was highest in pH inmice treated with compound D (FIG. 7).

TABLE 17 Cohorts Buffer formulation Total dose/day Group E 7.5% SodiumCarbonate 300 μl Group F 7.5% Sodium Carbonate w selected 300 μlpenetration enhancers) Group G 7.5% Sodium Carbonate, 300 μl 6% SodiumBicarbonate Group H 33.3% TRIS 300 μl

In a fourth study, a variation of the second study above was preformedwhere mice received a total dose of 300 μl/day of the formulations shownin Table 17. Pre-dosing urine levels from 4 mice was 5.73±0.02(mean±SEM). As shown in FIG. 8, all compounds were able to increaseurine pH significantly. Urine pH increases were observed after the 2ndcollection of urine towards the end of the study day. Compound F (7.5%Sodium Carbonate with modified penetration enhancers) consistentlyinduced the highest urine pH (FIG. 8).

In a fifth study, compound F (7.5% Sodium Carbonate with selectedpenetration enhancers) were tested in three doses of cohorts as follows:1504/day, 2004/day, and 3004/day. One group was treated with 200 mMsodium bicarbonate drinking water as a positive control. Theformulations used in this study are shown in Table 18.

TABLE 18 Buffer Formulations Tested Cohorts Buffer formulation Totaldose/day Group F 7.5% Sodium Carbonate 150 μl Group F 7.5% SodiumCarbonate (w 200 μl selected penetration enhancers) Group F 7.5% SodiumCarbonate, 300 μl 6% Sodium Bicarbonate Group SB 200 mM SodiumBicarbonate water ad libitum

Pre-dosing urine from 6 mice was 5.6±0.03 (mean±SEM). The pH measurementresults are presented in FIG. 8, which also shows that the response ofcompound F at a dose of 150 μL/day was similar to that at 300 μL/day(FIG. 8)

In another study, three doses of compound F (7.5% Sodium Carbonate withselected penetration enhancers) were test in three of the cohorts304/day, 754/day, and 1504/day. One group was treated with 200 mM sodiumbicarbonate drinking water as a positive control. The formulationstested are shown in Table 19.

TABLE 19 Buffer Formulations Tested Cohorts Buffer formulation Totaldose/day Group F 7.5% Sodium Carbonate  30 μl Group F 7.5% SodiumCarbonate (w  75 μl selected penetration enhancers) Group F 7.5% SodiumCarbonate, 150 μl 6% Sodium Bicarbonate Group SB 200 mM SodiumBicarbonate water ad libitum

The pre-dosing urine pH from 3 mice was 5.57±0.06 (mean±SEM). As shownin FIG. 9, urine pH values trended lower in all groups in this round ofstudies. Higher urine pH levels were observed in the 150 μL/day cohort,and was most similar to the sodium bicarbonate water positive controlgroup (FIG. 9).

In another study, three doses of compound F (7.5% Sodium Carbonate) wastested in three of the cohorts at doses of 1004/day, 1254/day, and1504/day. One group was treated with 200 mM sodium bicarbonate drinkingwater as a positive control. The formulations tested are shown in Table20 and results of the dose response curve are shown in FIG. 10. Withreference to the selected penetration enhancers referenced in theformulations of Table 19 and 20, these were 0.11% diethanolamine, 1.94%sodium caprate, and 0.20% sodium lauryl sulfate. EDTA in an amount of0.30% was also added to the formulations of Table 19 and 20. Otherpenetration enhancers as well as different amount of penetrationenhancers are suitable in other embodiments.

TABLE 20 Buffer Formulations Tested Cohorts Buffer formulation Totaldose/day Group F 7.5% Sodium Carbonate 100 μl Group F 7.5% SodiumCarbonate (w 125 μl selected penetration enhancers) Group F 7.5% SodiumCarbonate, 150 μl 6% Sodium Bicarbonate Group SB 200 mM SodiumBicarbonate water ad libitum

FIG. 10 is a dose response curve showing urine pH for 3 days, showingthe 200 mM sodium bicarbonate control, 100 μl of formulation, 125 μl offormulation, and 150 μl of formulation.

FIG. 11 shows urine pH measured over 3 days for two pH modulatingbuffering formulations: A, B, and 200 mM sodium bicarbonate in wateradministered as libitum as a control. These pH modulating bufferingcompounds are presented in Table 21.

TABLE 21 Formulation Compositions Study Ingredient Formula A Formula BLIP 14.00% 15.00% BA  1.00%  1.00% Menthol  0.25%  0.25% Durasoft  1.50% 1.50% Pluronic Granules  5.40%  2.10% Water 31.60% 29.65% SodiumCarbonate — — Sodium Bicarbonate 32.50% 32.50% EGTA — — sodium deconate— — propylene glycol  6.00% 10.00% almond oil  4.00%  3.00% zinc oxide 0.25%  0.50% cetyl alcohol  2.00%  3.00% ethanol  1.50%  1.50%

FIG. 12 shows urine pH measured over 3 days for three pH modulatingbuffering formulations: A, #25, #28, #29 and 200 mM sodium bicarbonatein water administered as libitum as a control. These pH modulatingbuffering compounds are presented in Table 20.

TABLE 20 Formulation Compositions Study Ingredient Formula 25 Formula 28Formula 29 LIP 6.00% 12.00%  12.00%  BA 1.00% 1.00% 1.00% Menthol 0.50%0.50% 0.50% Pluronic Granules 4.20% 4.20% 4.20% Water 37.80%  37.80% 40.30%  Sodium Carbonate 7.00% 7.00% 7.00% Sodium Bicarbonate 28.00% 28.00%  28.00%  EGTA — — 0.50% propylene glycol 3.00% 3.00% — almond oil3.00% 3.00% 3.00% cetyl alcohol 3.00% 3.00% 3.00% lecithin 3.00% — —cetiol ultimate 3.00% — — ethanol 1.50% 1.50% 1.50%

FIG. 13 shows urine pH measured over 3 days for three pH modulatingbuffering formulations: #25, #28, and #29 and control (SB, 200 mM sodiumbicarbonate in water).

FIG. 14 shows urine pH measured over 3 days for four pH modulatingbuffering formulations: #22A, #22B, #23, and #24.

The data and results from the studies reported in Example 6 can besummarized as follows. A formulation of 150 μL/day of 29.0% Lysine HCL,9.3% Trisodium Phosphate, and 23.4% TRIS did not significantly raiseurine pH. Doubling the doses (300 μL/day) of 9.3% Trisodium Phosphateand 23.4% TRIS did not significantly raise urine pH. Doubling dose (300μL/day) of 29.0% Lysine had no impact on urine pH. All formulations ofsodium carbonate tested consistently increases urine pH. A formulationof 15.8% sodium carbonate is highly effective in raising urine pH, butcaused skin irritation marked by redness and scabbing within 3 days. Aformulation of 7.5% sodium carbonate (with selected penetrationenhancers) achieved similar outcomes as 15.8%, but with no skinirritation. A formulation of 7.5% sodium carbonate (with modifiedpenetration enhancers) appeared to have a greater impact on increasingurine pH than 7.5% sodium carbonate. Changes in urine pH induced byformulations comprising 7.5% sodium carbonate (with selected penetrationenhancers) appears to be dose dependent. Doses of 7.5% sodium carbonate(with selected penetration enhancers) higher than 150 μL/day were notany more effective in increasing urine pH. The dose of 150 μL/day of7.5% sodium carbonate (with selected penetration enhancers describedherein) was more optimal in raising urine pH than lower doses. Urine pHincreased after application during the day, then returned to baselinelevels overnight.

Example 7—CPE Formulation

This is an example of an integrative cooperative CPE formulationdirected to the extra-cellular matrix to which might be added selectedcysteine cathepsin protease-inhibitors, with or without a suitablebuffering agent to NHE1 isoform inhibitor as a synergistic composition.

1. Cetyltrimethyl ammonium bromide (from about 2.0% to about 10.0%)

2. Sodium cholate: Lecithin (96% pure): Isopropyl myristate (equi-molar1:1:1 (from about 10% to about 40.0%)

3. Sodium citrate (titrate to transparency/incr. viscosity of #2.)

4. Benzyl alcohol (from about 2.0% to about 30.0%)

5. Cis-Palmitoleic acid (from about 20.0% to about 30% of BA)

6. Methyl pyrrolidone (0.4%)/Dodecyl pyridinium (1.1%) (from about 0.5%to about 5.0%)

7. Pluronic 127 (qs to 100%)

Example 8—Penetration Enhancing Formulation

This is an example of the formulation, which is directed to the cellularcomponent of the SC permeability barrier to which might be addedselected cysteine cathepsin protease-inhibitors, with or without asuitable buffering agent, and NHE1 isoform inhibitor as a synergisticcomposition.

1. ACSSSPSKHCG,[alanine-cysteine-serine-serine-serine-proline-serine-lysine-hisitidine-cysteine-glycine]identified as TD-1

2. Thioglycolic Acid (TGA) (from about 2.0% to about 7.0% concentration)[may be substituted by other reducing agents]

3. Proteinase K (from about 5 mg/mL to about 15 mg/mL)

Example 9—Use of Topical Buffering Agents to Increase Effectiveness ofWeak Base Chemotherapeutics without Adverse Events

In this experiment, doxorubicin in conjunction with a formulations ofthe invention are tested for their ability to enhance the cytotoxicitywithout adverse events observed with buffering therapies areadministered orally.

In vitro test were first performed and demonstrated significantincreases in drug uptake and cytotoxicity. Experiments were performedusing MCF-7 cells grown to log phase in 96-well plates and medium wasexchanged for one at either pH 6.8 or 7.4 containing 0.208 μCi per wellof 14C doxorubicin. Medium pH was buffered using non-volatile buffers(10 mM MES, 20 mM HEPES and 10 mM TRICINE) in combination withbicarbonate concentrations that were adjusted to be in equilibrium with5% ambient carbon dioxide. Twenty-four hours later, regular growthmedium was replaced and cells allowed to grow a subsequent 72 h, afterwhich time they were fixed and stained with crystal violet fordetermination of cell number. In-vitro, raising the pHe from 6.8 to 7.4resulted in a 2.25-fold enhancement of cytotoxicity and a 2.56-foldincrease in intracellular doxorubicin concentrations.

In vivo tests were performed and doxorubicin in conjunction withbuffering formulations of the invention demonstrated a significantlylowered tumor growth rate compared to treatment with doxorubicin aloneand without adverse events observed when buffering therapies wereadministered orally. In this experiment MCF-7 tumors were grown in themammary fat pads of 6-week-old female SCID mice, to sizes of 50-200 mm3,and were forcibly randomized according to tumor size into six groups:

-   -   Group A and B: Control    -   Group C and D: 200 mM sodium bicarbonate drinking water ad        libitum    -   Group E and F: 504,×3 doses approximately Q8 hours (total daily        dose of 150 μL) of formulation in Table 21 below as follows:

TABLE 21 Group E and F Ingredient Formulation LIP 6.00% BA 1.00% Menthol0.50% Pluronic Granules 4.20% Water Q.S. Sodium Bicarbonate 33.00% Propylene glycol 3.00% almond oil 3.00% cetyl alcohol 3.00% lecithin3.00% cetiol ultimate 3.00% ethanol 1.50%

The start of the bicarbonate treatment was designated as day 1. On days3, 7 and 11, animals in groups B, D, and F were injected i.p. with 1.6mg kg-1 doxorubicin while animals in groups A, C, and E (n=10 each)received saline injections of the same volume, as per establishedprotocols. On day 15, animals in Groups C and D were placed back onnormal drinking water and animals in Groups E and F ceased receivedt.i.d. topical applications.

Tumor volumes and animal weights were monitored every 2 days.Doxorubicin had a significant effect on the tumor growth rate (Groups Bgrowth rate decreased ˜30% over assessment period). This effect wasgreater in animals co-treated with bicarbonate whether by oral ortopical administration (With both Groups D and F observing a 55%reduction in growth rate). In this embodiment, bicarbonate alone (GroupsC and E) had no significant effect on the growth rate alone. Othercharacteristics of tumors that increase its potential for carcinogenesisother than growth rates are measured by other parameters as well,including inhibition metastasis. In the inventor's experience, differentembodiments disclosed herein may have differential effects on differentcharacteristics of tumors (e.g. growth rate, volume, metastasis, pHmicroenvironment, etc.).

In addition to tumor volumes, animals in group D experienced analdischarge suggesting increased GI tract cytotoxicity of doxorubicin whenorally dosed. This adverse event was not noted in topically dosedanimals that received doxorubicin (Group F).

Example 10—Use of Topical Buffering Agents to Increase Effectiveness ofImmunotherapies in Melanoma

In this experiment, we studied the effects of a formulations of theinvention on the efficacy of immunotherapy for melanoma. It isunderstood that the highly acidic tumor microenvironment might blunt theeffectiveness of anti-tumor immunity.

For three days C57BL/6 mice received 50 μL×3 doses approximately QShours (total daily dose of 1504) of 33% sodium bicarbonate in aformulation of the invention detailed below. On day 4, the mice weresubject to tumor injection and continued to receive the sodiumbicarbonate topical until the end of the experiment. Control micereceived a placebo topical cream as detailed in Table 22 below.

TABLE 22 Ingredient Control Active Ethanol 1.50% 1.50% Benzyl Alcohol1.00% 1.00% Cetyl Alcohol 2.00% 2.00% Almond Oil 3.00% 3.00% Lecithin10.00%  7.00% IPP 10.00%  7.00% Propylene Glycol 5.00% 5.00%Poloxamer/Plumnic Powder 9.00% 5.40% Water Q.S. Q.S. Sodium Bicarbonate0.00% 33.00%  Durosoft PK-SG 1.00% 1.00% 100.00%  100.00% 

A total of 1×10⁵ B16 melanoma tumor cells were injected s.c. in the leftflank of the C57BL/6 mice. Mice received i.p. injections of 20 mg/kg ofanti-PD1 or anti-CTLA4 antibodies on day 4 and continued to receiveantibodies every 3 days until the end of the experiment. Mice werehumanely euthanized when tumors exceeded 1.5 cm in diameter, appearednecrotic, or interfered with locomotion. Tumors were collected andweighed.

The topical sodium bicarbonate improved CTLA4 therapy in B16 melanomadecreasing mean tumor size from ˜800 mm³ in controls to ˜550 mm³ inactives and decreasing tumor weight from a mean of ˜775 mg in thecontrol arm to a mean of ˜400 mg in the active arm.

The topical sodium bicarbonate improved PD1 therapy in B16 melanomadecreasing mean tumor size from ˜250 mm² in controls to ˜150 mm² inactives and decreasing tumor weight from a mean of ˜650 mg in thecontrol arm to a mean of ˜325 g in the active arm.

Example 11=Use of Topical Buffering to Increase Effectiveness ofImmunotherapies in Pancreatic Tumors

In this experiment, we studied the effects of formulations of theinvention on the efficacy of immunotherapy for pancreatic tumors. It isunderstood that the highly acidic tumor microenvironment might blunt theeffectiveness of anti-tumor immunity.

For three days C57BL/6 mice received 50 μL×3 doses approximately Q8hours (total daily dose of 150 μL) of 33% sodium bicarbonate in aformulation of the invention detailed below. On day 4, the mice weresubject to tumor injection and continued to receive the sodiumbicarbonate topical until the end of the experiment. Control micereceived a placebo topical cream as detailed in Table 23 below.

TABLE 23 Ingredient Control Active Ethanol 1.50% 1.50% Benzyl Alcohol1.00% 1.00% Cetyl Alcohol 2.00% 2.00% Almond Oil 3.00% 3.00% Lecithin10.00%  7.00% IPP 10.00%  7.00% Propylene Glycol 5.00% 5.00%Poloxamer/Pluronic Powder 9.00% 5,40% Water Q.S. Q.S. Sodium Bicarbonate0.00% 33.00%  Durosoft PK-SG 1.00% 1.00% 100.00%  100.00% 

A total of 1×10⁵ Panc02 pancreatic tumor cells were injected s.c. in theleft flank of the C57BL/6 mice. Mice received i.p. injections of 20mg/kg of anti-PD1 antibodies on day 4 and continued to receiveantibodies every 3 days until the end of the experiment. Mice werehumanely euthanized when tumors exceeded 1.5 cm in diameter, appearednecrotic, or interfered with locomotion. Tumors were collected andweighed.

The topical sodium bicarbonate improved PD1 therapy in Panc02 decreasingtumor weight from a mean of ˜650 mg in the control arm to a mean of ˜300g in the active arm.

Example 12—Use of Topical Buffering Agents to Decrease Primary TumorMetastases and Increase Survival in Metastatic Breast Cancer

In this experiment, topical applications of buffer with formulations ofthe invention were tested for their ability to buffer extracellularacidity and inhibit the spread of metastases and increase overallsurvival in a mouse model for metastatic breast cancer. The topicalformulations of the invention were compared to a “no treatment” controlas well as orally delivered buffer as a positive control.

In vivo tests were performed as follows: 72 female Ncr nude mice aged 6weeks were injected with 5×10⁶ MDA-MB-231/eGFP cells in the mammaryfatpad to generate orthotopic “primary” tumors. The following day aftertumor inoculation, mice were then randomized into 5 treatment groups asoutlined below.

The treatment groups were:

Group A: Untreated Control

Group B: 200 mM sodium bicarbonate drinking water ad libitum

Group C: 50 μL×3 doses daily (total daily dose of 150 μL) of formulationdetailed below

Group D: 50 μL×3 doses daily (total daily dose of 150 μL) of formulationdetailed below

Group E: 50 μL×3 doses daily (total daily dose of 150 μL) of formulationdetailed below

The formulations of the embodiment are shown in Table 24 were asfollows:

TABLE 24 Ingredient % weight Group C Menthol 0.50% Ethanol 1.50% BenzylAlcohol 1.00% Cetyl Alcohol 2.00% Almond Oil 3.00% LIP 14.00%  PropyleneGlycol 5.00% 30% Pluronic Gel 18.00%  DI Water 21.00%  SodiumBicarbonate 33.00%  Durosoft PK-SG 1.00% Total 100.00%  Group D Menthol0.50% Benzyl Alcohol 1.00% LIP 25.00%  Cetyl Alcohol 1.50% Stearic Acid1.50% Deionized Water 3.24% Ethanol 1.50% 30% Pluronic Gel 30.56%  NaOH50% Solution 1.00% Sodium Bicarbonate 33.20%  Durosoft PK-SG 1.00% Total100.00%  Group E Menthol 0.50% Benzyl Alcohol 1.00% Cetyl Alcohol 2.00%Stearic Acid 2.00% Almond Oil 3.00% LIP 14.00%  Ethanol 1.50% PropyleneGlycol 5.00% 30% Pluronic Gel 18.00%  DI Water 18.00%  50% NaOH Solution1.00% Sodium Bicarbonate 33.00%  Durosoft PK-SG 1.00% Total 100.00% 

Application of transdermal agent in treatment groups C, D, and Eoccurred 3 times/day for 120 days. Volumes of primary tumors in mammaryfat pads were measured twice weekly and calculated from orthogonalmeasurements of external dimensions as (width)2×(length)/2. Surgicalresections of primary tumors occurred tumors reached 350-500 mm3. Micewere euthanized by cervical dislocation when tumor burden becameexcessive (primary, intraperitoneal, or lymph node >2000 mm3) or whenmouse progressed to a moribund state. Survival data were expressed as aKaplan-Meier curve.

Upon termination of the survival experiment, tumor metastases wereidentified by gross necropsy. All tumor tissue was fixed in 10% neutralbuffered formalin (NBF). The green fluorescent (GFP) tumors weredetected using a 470 nm/40 nm excitation filter and imaged using amounted digital camera. Whole lung images data were analyzed with AdobePhotoshop 5.0 using the “magic wand” tool to select lung area and greenfluorescent tumor lesions. Pixel area of the selected images wasmeasured using ImageJ.

The primary tumor growth rates were observed to be the same in allactive Groups, Groups B, C, D, and E. This is consistent with earlierfindings in orally administered buffering therapies.

There were significant differences observed in metastatic rates in allgroups treated with buffer, Groups B, C, D, and E. In addition,topically applied buffer groups, Groups C, D, and E demonstrated lowermetastatic rates compared to orally dosed, Group B. As follows:

In the untreated Group A, metastatic rates were as follows:

Intestinal: 36%

Mesentery: 14%

Lymph Node: 64%

Lung: 79%

In the orally treated, Group B, metastatic rates were as follows:

Group A Metastatic Rates:

Intestinal: 0%

Mesentery: 0%

Lymph Node: 27%

Lung: 8%

In the topically treated groups, Group C, D and E metastatic rates wereall lower and within the following ranges:

Group A Metastatic Rates:

Intestinal: 0%

Mesentery: 0%

Lymph Node: 5-27%

Lung: 3-8%

There were significant differences observed in survival rates in allgroups treated with buffer, Groups B, C, D, and E. In addition,topically applied buffer groups, Groups C, D, and E demonstrated lowermetastatic rates compared to orally dosed, Group B. As follows:

% of mice surviving to 120 days:

Group A: 20%

Group B: 60%

Groups C-G: 60%-70%

Example 13—Treatment Primary Tumor Growth, pH and Spontaneous Metastaseswith Formulations of the Invention

To investigate the effect of various formulations provided herein onprimary tumor growth, Luciferase expressing prostate cancer cells,PC3M-luc, will be subcutaneously into the right flank of male SCID mice.A suitable skin portion of all animals can be removed of hair to allowdirected application of a particular formulation. Four days prior toinjections, half of the animals will be treated with different amountsof selected formulation.

Animals will be imaged weekly via bioluminescence. Images fromrepresentative animals will be taken as an indication of growth of theprimary tumor in both cohorts of animals. A plot of bioluminescence ofthe primary tumor versus time can then be established.

The tumor pH will also be taken immediately prior to euthanasia (29 daysafter injection of the primary tumor), and the tumors are to beharvested immediately afterwards. The mean tumor pH for each animal willbe averaged within each treatment group (tap versus IEPA); and the errorcan be calculated as the standard error between the mean tumor pH ineach group.

Necrosis of the primary tumor can also be studied by H&E staining andcounted by area, as outlined in Hashim et al., Clin Exp. Metastasis(2011), 28:841-849; DOI 10.1007/s10585-011-9415-7, incorporated byreference herein.

Methods

Three days prior to inoculation with tumor cells, 4-6 week old malebeige SCID mice (Harlan, Madison, Wis.) are placed into experimentalcohort groups. Animal weights were measured and recorded twice weekly,and the overall health of each animal was noted to ensure timelyendpoints within the experiment.

Cell culture and inoculation. PC3M cells (-Luc6 clone) are availablefrom obtained from Caliper (Hopkinton, Mass.). The cells are culturedusing MEM/EBSS media, supplemented with 10% fetal bovine serum, 1%penicillin streptomycin, 1% nonessential amino acids, 1% sodium pyruvateand 1% MEM vitamins. In preparation for inoculation into mice, the cellscan be trypsinized and rinsed once with sterile phosphate bufferedsaline (PBS) prior to resuspension at a concentration of 5×106 cells in200 μl PBS. For primary tumor injection, animals are prepared byremoving the hair from the injection site, and 200 μl containing 5×106cells in PBS were injected subcutaneously into the right flank of eachmouse. For experimental metastases, 200 μl containing 5×106 cells in PBSare then injected directly and slowly (over the course of 1 min) intothe tail vein of each mouse. In both preparations, cell distributionswere verified by bioluminescent imaging immediately following injection.

Bioluminescent imaging. Animals are anesthetized with isoflurane andinjected intraperitoneally with 10 μl per g body weight of steriled-luciferin substrate prepared in PBS at 15 mg/ml (resulting dose 150μg/g body weight). After 5 min, mice are transferred to thethermo-regulated, light-tight chamber of the In Vivo Imaging System,IVIS-200 (Caliper; Hopkinton, Mass.). Photographic images are acquiredfirst, and the bioluminescent images can be overlaid on top of theseimages. Bioluminescent images are acquired by measuring photons emittedfrom luciferase-expressing cells and transmitted through the tissue. Theexposure time for the bioluminescent image acquisition typically rangesfrom 0.5 s (whole tumor images) up to 2 min (lung metastases) to ensurenon-saturation, and differences in exposure time are corrected byexpressing data as total flux in photons/sec, rather than photon counts.Images can be analyzed using the LivingImage software (Caliper;Hopkinton, Mass.)

Necrosis counting. The center section (˜5 mm) of the subcutaneous tumorof each animal will be fixed in paraffin blocks prior to staining one 4μm thick cross-sectional sample per animal with hematoxylin and eosinfor histology. Histology slides can be scanned using the Aperio™ (Vista,Calif.) ScanScope XT with a 20×/0.8 NA objective lens (200×) at a rateof 2 min per slide via Basler tri-linear-array. Image analysis can beperformed using an Aperio Genie® v1 customized algorithm in conjunctionwith Positive Pixel Count v9 with the following optimized thresholds[Hue value=0.2; Hue width=0.6; color saturation threshold=0.05;IWP(High)=210; Iwp(Low)=Ip(High)=160; Ip(low)=Isp(High)=80; Isp(Low)=0].The algorithm is applied to the entire slide's digital image todetermine the percentage of necrosis by detecting the number of pixelsthat satisfy the color and intensity specification defined above(necrotic), divided by the number of pixels in non-necrotic tissue.

Magnetic resonance imaging and spectroscopy. MR images and spectra canbe obtained on a Varian MR imaging spectrometer ASR310 (Agilent LifeSciences Technologies, Santa Clara, Calif.) with a 30 cm horizontalclear bore operating at a field strength of 7 T. For reference, ahigh-resolution spectrum of IEPA in D2O can be obtained on a VarianNuclear Magnetic Resonance spectrometer with a 54 mm vertical boreopening at a field strength of 9.4 T. For in vivo spectroscopic imaging,naïve mice were allowed to drink IEPA for 3 days prior to imaging. Theanimals are to be sedated using isoflurane, placed in the animal cradlefor insertion into the bore of the 7 T Varian MRI and maintained warmusing a continuous warm air blower (Small Animal Instruments, Inc.,Stonybrook, N.Y.). Temperatures can be measured using a fiber opticendorectal thermometer in conjunction with the MR compatible animalmonitoring system (Model 1025, Small Animal Instruments, Inc. StonyBrook, N.Y.). SCOUT images are taken to verify location, and T2 weightedimages for anatomical identification were obtained using a fast spinecho (FSEMS) pulse sequence, with FOV=40×80 (mm), 15 coronal slices, 1mm thick, no gap, TR=2450 s, effective TE=72 ms, with fat suppressionon. Spectra can be obtained using a stimulated echo (STEAM) localizationsequence on a 2×2×2.5 mm3 voxel in the bladder with 256 averages (flipangle 90 deg, TE 9.44, TM 8.01, and TR 2000), for an 8.5 minacquisition. Images and spectra were processed using the Varian Vnmrjsoftware or using MATLAB (MathWorks, Inc, Natick, Mass.).

Electrode measurement of pH. Animals can be sedated using isoflurane,and placed on a warming surface to maintain appropriate body temperaturefor the duration of the experiment. Both the needle microelectrode andthe reference electrode can be obtained from Microelectrodes, Inc.,(Bedford, N.H.). A shallow small (<5 mm) incision is typically made inan alternate (non tumor) site and the 1 mm reference electrode wasplaced subcutaneously therein. A needle micro electrode (OD 0.8 mm witha beveled end) is inserted up to 1.3 cm into the center of the tumor,and was held in place for up to 1 min, until pH readings stabilized. Theneedle is rotated once in each location, to allow the pH electrode tore-read at the same depth in order to make two independent measurementsper location. The pH is typically measured at three locations, one nearthe center/core of the tumor, one in a mid-region of the tumor, and oneat the rim of the tumor; these values are averaged to report a mean foreach animal. After the pH was measured in the primary tumor, the animalsare euthanized (29 days after subcutaneous injection of the primarytumor cells). Before and after the pH is measured in each animal, the pHelectrodes are used to measure a standard pH 7 buffer solution (ThermoFisher Scientific, Inc., Waltham, Mass.).

Example 14—Use of Formulations of the Invention on Syngenic MelanomaGrowth in CB57/BL Mice

In this experiment serval formulations according to the invention areevaluated for effectiveness on tumor growth in a syngenic melanoma mousemodel.

In vitro study. The murine B16F10 and the human A375 melanoma cell lineis available from ATCC (Milan, Italy); the Me1501, Me30966 and WM793human melanoma cells are also available commercially. Cells are seededin 96 wells plates at the concentration of 5000 cells/well. The cellsare maintained in RPMI culture medium with 10% of FBS at pH 7.4.

In vivo studies. Approximately 5×105 B16F10 melanoma cells aresubcutaneously injected in the right flank of subject animals. Tumorsare calipered twice a week and mice were weighted once a week. Mice aredivided into a control group and treatment groups that receive differentdosages of various formulations provided herein. Mice are checked twicea week by a veterinarian responsible for animal welfare monitoring forsigns of sufferance such as weight loss, decreased water and foodconsumption, poor hair coat, decreased activity levels and tumorulcerations. Endpoints were maximum tumor volume of 1200 mm3 accordinglyto the guidelines for a correct laboratory practice and signs of poorquality of life.

In vivo MRI guided 31P MRS. A group of CB57/BL mice carrying syngenicmelanoma tumors B16F10 implanted in the right flank are subjected to anMRI study. Upon reaching the tumor volume of 800 mm3 mice were gavagedwith a single non-toxic dose of 4 or 8 g/l of formulation solubilized in200 μl of water. Mice are subjected to MRI/MRS analyses by using aVarian Inova 200/183 MRI/MRS system for small animals operating at 4.7T. Animals can be anesthetized with sevoflurane 2.5% in O2 11/min.Throughout the anesthesia procedures the ECG, PO2, and PCO2, are to beroutinely monitored as per ISS guidelines and current literature.Temperature is maintained at 37±0.5° C. by a feedback controlled watercirculating heating cradle. Tumor extracellular pH (pHe) value ismeasured from chemical shift difference between the exogenous cellimpermeant 31P reporter 3-APP resonance and that of α-ATP.

The 3-APP probe (128 mg/kg) is administered i.p. immediately prior toMRI/MRS analyses. A three turn 31P surface coil specifically designed tofit superficial tumors combined with a butterfly 1H coil (RAPIDBiomedical, Rimpar, Germany) for shimming and positioning of the volumeof interest (VOI) can be used. T1-weighted gradient-echo multislicecontiguous images (TR/TE=400/3.5 msec, a α 70°, thickness=1 mm, 8averages, 19 slices, matrix 128×128, FOV=3×3 cm2 which correspond to inplane resolution of 0.2×0.2 mm2) are acquired to localize the tumor. 1Hlocalized spectra is used to optimize magnetic field homogeneity inorder to increase the signal resolution within the tumor (1H PRESS,TR/TE=2,000/23 msec). 31P localized spectra are acquired from the tumorwith a pulse-acquire sequence (TR=3,000 ms, α=25°, 256 averages) beforeand up to 1.5 h after formulation administration.

Statistical analysis. Differences between treatment groups, both invitro and in vivo, can be analyzed by one-way ANOVA and Bonferronit-test. Data can be expressed as mean±SD and SE for in vivo experiments,and p values reported are two-sided. P values <0.05 are generallyconsidered as statistically significant. Statistical analysis can beperformed with Sigmastat 2006 software.

Example 15—Coadministration Treatments and Combinational Therapies

In this experiment, a more exhaustive study of several differentchemotherapeutic agents, immunotherapeutic agents, and biological agentswill be performed. Various biological, chemotherapeutic, andimmunotherapeutic agents are tested in conjunction with formulations ofthe invention for their ability to treat cancer, reduce metastasis,reduce carcinogenesis, maintain remission, and the other methodsrelating to cancer and tumors described herein. Methods are performed inaccordance with Examples 9-11 above, with particular biologic,chemotherapeutic and immunotherapeutic agents additionally added to testtheir effect on primary tumor growth.

Biological agents that are cytokines such as interferons andinterleukins will be tested.

Also tested will be chemotherapeutic agents such as alkylating agents,anthracyclines, antimetabolites, antitumor antibiotics, aromataseinhibitors, taxanes and related compounds, cytoskeletal disruptors,epothilones, histone deacetylace inhibitors, kinase inhibitors,nucleoside analogues, topoisomerase inhibitors, retinoids, and vincaalkaloids and derivatives thereof. Chemotherapeutic agents to be testedinclude anthracyclines, antimetabolites, antitumor antibiotics,aromatase inhibitors, taxanes, and others described herein.

Immunotherapeutic agents include antibodies and cell based approacheswill be tested. Those tested will include alemtuzumab, atezolizumab,avelumab, ipilimumab, durvalumab, nivolumab, ofatumumab, rituximab, andothers described herein.

In related experiments, in vivo approaches to modifying gene expressionare tested in conjunction with formulations of the invention for theirability to reduce cancer, metastasis, inhibit or prevent carcinogenesis,enhance immune response, etc. One approach tested will utilize theCRISPR to modify genes and gene expression of selected target genesdescribed herein and to use this therapy in conjunction with certaintopical formulations of the invention for various aspects of treatingcancer described herein such as treating or preventing cancer,preventing carcinogenesis, maintaining remission, and the like. Suitablemethods for screening targets and performing CRISP are described in U.S.Ser. No. 15/575,325 entitled ‘Screening Methods for Cancer Therapy’,filed Nov. 17, 2017, incorporated by reference herein.

Aspects of the present specification may also be described as follows:

1. A method of treating a proliferative disorder associated with cancerin a patient, the method comprising administering an effective amount ofa formulation for transdermal delivery through the skin of a subjectcomprising one or more buffering agent to a patient in need thereof,wherein said administration is effective to i) inhibit or prevent thegrowth of a tumor or tumor cells, ii) inhibit or prevent the metastasisof tumors or cancer cells, iii) inhibit or prevent carcinogenesis, iv)inhibit or prevent the intravasation of tumor cells, or v) improve orextend the duration of remission, or maintain remission of a cancer ortumor.

2. A method according to claim 1, wherein said treating a proliferativedisorder inhibits or prevents the growth of a tumor or tumor cells.

3. A method according to claim 1, wherein said treating a proliferativedisorder inhibits or prevents the metastasis of tumors or cancer cells.

4. A method according to claim 1, wherein said treating a proliferativedisorder inhibits or prevents carcinogenesis.

5. A method according to claim 1, wherein said treating a proliferativedisorder inhibits or prevents the intravasation of tumor cells.

6. A method according to claim 1, wherein said treating a proliferativeimproves or extends the duration of remission or maintains remission ofa cancer or tumor.

7. A method of treating cancer in a patient, the method comprisingadministering an effective amount of a formulation for transdermaldelivery through the skin of a subject comprising one or more bufferingagent to a patient in need thereof, wherein said administration iseffective to inhibit or prevent the growth of a tumor or tumor cells.

8. A method of preventing metastasis of tumors, the method comprisingadministering an effective amount of a formulation for transdermaldelivery through the skin of a subject comprising one or more bufferingagent to a patient in need thereof, wherein said administration iseffective to inhibit or prevent the metastasis of tumors or cancercells.

9. A method according to claim 1, wherein said formulation fortransdermal delivery through the skin of a subject comprises a bufferingagent comprising a carbonate salt in an amount between about 10-56% w/w;a penetrant portion in an amount between about 5 to 55% w/w; a detergentportion in an amount of at least 1% w/w; and wherein the formulationcomprises water in an amount from 0% w/w up to 70% w/w, and wherein theformulation optionally comprises lecithin in an amount less than about12% w/w.

10. A method according to claim 1, wherein said formulation fortransdermal delivery through the skin of a subject comprises a bufferingagent comprising at least one carbonate salt, lysine, tris, a phosphatebuffer and/or 2-imidazole-1-yl-3-ethoxycarbonylpropionic acid (IEPA), ora combination thereof in an amount between about 10-56% w/w; and apenetrant portion in an amount between about 44 to 90% w/w, wherein thepenetrant portion comprises water in an amount less than about 85% w/w,and wherein the formulation comprises less than about 12% w/w lecithin.

11. A method according to claim 10, wherein a chemotherapeutic orimmunotherapeutic agent is co-administered with said formulationcomprising one or more buffering agent.

12. A method according to claim 10, wherein said administration iseffective to alter the pH of a tissue or microenvironment proximal to asolid tumor or cancer cells in the patient.

13. A method according to claim 11, wherein the chemotherapeutic orimmunotherapeutic agent is selected from alkylating agents, antibodiesand related binding proteins, anthracyclines, antimetabolites, antitumorantibiotics, aromatase inhibitors, taxanes and related compounds,cytoskeletal disruptors, epothilones, histone deacetylace inhibitors,kinase inhibitors, nucleoside analogues, topoisomerase inhibitors,retinoids, and vinca alkaloids and derivatives thereof.

14. A method according to claim 13, wherein the chemotherapeutic orimmunotherapeutic agent is an immunotherapeutic agent selected fromalemtuzumab, atezolizumab, avelumab, ipilimumab, durvalumab, nivolumab,ofatumumab, rituximab and trastuzumab.

15. A method according to claim 10, comprising a carbonate salt in anamount between about 7-56% w/w of the formulation.

16. A method according to claim 9 or 15, wherein the carbonate salt insaid formulation is in an amount between about 15-32% w/w of theformulation.

17. A method according to claim 9 or 15, wherein the carbonate salt insaid formulation is sodium carbonate and/or sodium bicarbonate milled toa particle size is less than 200 μm.

18. A method according to claim 9 or 15, wherein the penetrant componentin said formulation is in an amount between about 18-42% w/w of theformulation.

19. A method according to claim 9 or 15, wherein the water in saidformulation is in an amount between about 15-42% w/w of the formulation.

20. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises an alcohol in an amount less than 5% w/wof the formulation.

21. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises lecithin organogel, an alcohol, asurfactant, and a polar solvent.

22. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises lecithin organogel in an amount less than5% w/w of the formulation.

23. A method according to claim 22, wherein the lecithin organogel insaid formulation is a combination of soy lecithin and isopropylpalmitate.

24. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises lecithin and isopropyl palmitate,undecane, isododecane, isopropyl stearate, or a combination thereof.

25. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises a mixture of xanthan gum, lecithin,sclerotium gum, pullulan, or a combination thereof in an amount lessthan 5% w/w of the formulation.

26. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises a mixture of caprylic triglycerides andcapric triglycerides in amount less than 8% w/w of the formulation.

27. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises phosphatidyl choline in amount less than12% w/w of the formulation.

28. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises a phospholipid in amount less than 12% w/wof the formulation.

29. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises a mixture of tridecane and undecane inamount less than 5% w/w of the formulation.

30. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises cetyl alcohol in amount less than 5% w/wof the formulation.

31. A method according to claim 9 or 15, wherein the penetrant portionin said formulation comprises benzyl alcohol in an amount less thanabout 5 w/w.

32. A method according to claim 9 or 15 wherein the penetrant portion insaid formulation comprises stearic acid in an amount less than 5% w/w ofthe formulation.

33. A method according to claim 9 or 15, wherein said formulationcomprises a gelling agent in an amount less than 5% w/w of theformulation.

34. A method according to claim 9 or 15 wherein the detergent portion insaid formulation comprises a nonionic surfactant in an amount betweenabout 2-25% w/w of the formulation; and a polar solvent in an amountless than 5% w/w of the formulation.

35. A method according to claim 34, wherein the nonionic surfactant insaid formulation is a poloxamer and the polar solvent is water, analcohol, or a combination thereof.

36. A method according to claim 34, wherein the detergent portion insaid formulation comprises poloxamer, propylene glycol, glycerin,ethanol, 50% w/v sodium hydroxide solution, or a combination thereof.

37. A method according to claim 9 or 15, wherein the detergent portionin said formulation comprises glycerin in an amount less than 3% w/w ofthe formulation.

38. A method according to claim 9 or 15, wherein the carbonate salt issodium carbonate and/or sodium bicarbonate in said formulation is milledto a particle size is less than 70 μm.

39. A method according to claim 9 or 15, wherein the carbonate salt insaid formulation is sodium carbonate and/or sodium bicarbonate milled toa particle size is less than 70 μm, wherein the sodium bicarbonate issolubilized in the formulation in an amount less than 20% w/w of theformulation.

40. A method according to claim 9 or 15, wherein the carbonate salt insaid formulation is sodium carbonate and/or sodium bicarbonate milled toa particle size is less than 70 μm, wherein particle sizes less thanabout 10 μm have an enhanced penetration thru the skin of a subject.

41. A method according to claim 9 or 15, wherein said formulationfurther comprises tranexamic acid in an amount less than 5% w/w of theformulation.

42. A method according to claim 9 or 15, wherein said formulationfurther comprises a polar solvent in an amount less than 5% w/w of theformulation.

43. A method according to claim 9 or 15, wherein said formulationfurther comprises a humectant, an emulsifier, an emollient, or acombination thereof.

44. A method according to claim 9 or 15, wherein said formulationfurther comprises ethylene glycol tetraacetic acid in an amount lessthan about 5 w/w.

45. A method according to claim 9 or 15, wherein said formulationfurther comprises almond oil in an amount less than about 5 w/w.

46. A method according to claim 9 or 15, wherein said formulationfurther comprises a mixture of thermoplastic polyurethane andpolycarbonate in an amount less than about 5 w/w.

47. A method according to claim 9 or 15, wherein said formulationfurther comprises phosphatidylethanolamine in an amount less than about5% w/w.

48. A method according to claim 9 or 15, wherein said formulationfurther comprises an inositol phosphatide in an amount less than about 5w/w.

49. A method of preventing the intravasation of tumor cells, the methodcomprising administering topically and/or transdermally an effectiveamount of a formulation for transdermal delivery comprising one or morebuffering agent to a patient in need thereof, wherein saidadministration is effective to inhibit or prevent the intravasation oftumor cells.

50. A method of improving, extending the duration of remission, ormaintaining remission of a cancer or tumor, the method comprisingadministering topically and/or transdermally an effective amount of aformulation for transdermal delivery comprising one or more bufferingagent to a patient in need thereof, wherein said administration iseffective to improve or extend the duration of remission or maintainremission of a cancer or tumor.

51. A method of treatment of cancer comprising i) selecting atherapeutic agent comprising. a chemotherapeutic or an immunotherapeuticagent, ii) formulating the therapeutic agent in a suitable formulation,iii) administering the formulation comprising the therapeutic agent, andiv) before, during or after step iii), administering a formulation fortransdermal delivery comprising one or more buffering agent topicallyand/or transdermally in an amount effective to inhibit or prevent thegrowth of a tumor or tumor cells.

52. A method of altering the pH of a tissue or microenvironment proximalto a solid tumor or cancer cells in a patient, the method comprisingadministering topically and/or transdermally an effective amount of aformulation for transdermal delivery comprising one or more bufferingagent to a patient in need thereof, wherein said administration iseffective to alter the pH of a tissue or microenvironment proximal to asolid tumor or cancer cells in the patient.

53. A method of treating a disease or disorder associated with abnormallevels of uric acid in a patient, the method comprising administeringtopically and/or transdermally an effective amount of a pharmaceuticalformulation comprising one or more buffering agent to a patient havingabnormal levels of uric acid and in need thereof, wherein saidadministration is effective to treat or reduce the symptoms of abnormallevels of uric acid in said patient.

54. A method according to claim 53, wherein the disease or disorderincludes one of more of the following: gout, recurrent gout attack,prevention of gout, gouty arthritis, hyperuricaemia, gout-relatedcardiovascular disorders, Lesch-Nyhan syndrome, Kelley-Seegmillersyndrome, kidney disease, kidney stones, kidney failure, jointinflammation, inflammatory joint disease, arthritis, osteoarthritis,rheumatoid arthritis and psoriatic arthritis, urolithiasis, plumbism,hyperparathyroidism, psoriasis or sarcoidosis.

55. A method according to claim 54, wherein the disease or disorder isgout or the prevention of gout.

56. A method according to claim 54, wherein the disease or disordercomprises urolithiasis, kidney stones, or bladder stones.

57. A method according to claim 56, wherein the subject is an animalthat is treated for urinary or renal stones.

58. A method of treating a urinary stone in a patient, the methodcomprising administering topically and/or transdermally an effectiveamount of a pharmaceutical formulation comprising one or more bufferingagent to a patient in need thereof, wherein said administration iseffective to ameliorate, treat or reduce the symptoms, size, or severityof the urinary stone.

59. A method according to the claim 58, wherein the patient is ananimal.

60. A method according to the claim 59, wherein the animal is cat ordog.

61. A method according to the claim 58, wherein the urinary stone is abladder or kidney stone.

62. A method of treating a skin disorder in a subject, the methodcomprising administering topically and/or transdermally an effectiveamount of a formulation comprising one or more buffering agent to apatient having a skin disorder and in need thereof, wherein saidadministration is effective to treat or reduce the skin disordersymptoms in said patient.

63. A method according to the claim 62, wherein the skin disorder ismelasma and said administration is effective to treat or reduce themelasma symptoms in said patient.

64. A method according to the claim 63, wherein the skin disorder ispremature aging and the method prevents or inhibits collagen acylationin the skin of said patient.

65. A method of treating melasma comprising administering one or morebuffering agent with a sun protecting lotion or cream.

66. A method according to any one of the preceding claims wherein saidformulation for transdermal delivery comprises a buffering agentcomprising a carbonate salt in an amount between about 10-45% w/w; apenetrant portion in an amount between about 5 to 55% w/w; a detergentportion in an amount between about 1 to 15% w/w; and wherein theformulation comprises water in an amount between about 15 to 65% w/w,and wherein the formulation comprises less than about 12% w/w lecithin.

67. A method according to claim 65, wherein the administering isperformed topically by directly contacting the skin of said subject withthe formulation provided to said subject.

68. A method according to claim 66, wherein prior to application of theformulation skin of said patient is pretreated by abrasion,tape-stripping, microderm-abrasion, or microneedling 69. A medicalformulation kit, the kit comprising a lotion for administering topicallyand/or transdermally a formulation comprising a buffering agent andadministration directions that includes instructions for amounts and usefor a medical professional.

70. A method according to claim 58, wherein the carbonate salt in saidformulation is in an amount between about 7-32% w/w of the formulation.

71. A method according to claim 65, wherein the carbonate salt in saidformulation is in an amount between about 15-32% w/w of the formulation.

72. A method according to claim 65, wherein the penetrant component insaid formulation is in an amount between about 18-42% w/w of theformulation.

73. A method according to claim 65, wherein the water in saidformulation is in an amount between about 15-42% w/w of the formulation.

74. A method according to claim 65, wherein the penetrant portion insaid formulation comprises an alcohol in an amount less than 5% w/w ofthe formulation.

75. A method according to claim 65, wherein the penetrant portion insaid formulation comprises lecithin organogel, an alcohol, a surfactant,and a polar solvent.

76. A method according to claim 65, wherein the penetrant portion insaid formulation comprises lecithin organogel in an amount less than 5%w/w of the formulation.

77. A method according to claim 18, wherein the lecithin organogel insaid formulation is a combination of soy lecithin and isopropylpalmitate.

78. A method according to claim 65, wherein the penetrant portion insaid formulation comprises lecithin and isopropyl palmitate, undecane,isododecane, isopropyl stearate, or a combination thereof.

79. A method according to claim 65, wherein the penetrant portion insaid formulation comprises a mixture of xanthan gum, lecithin,sclerotium gum, pullulan, or a combination thereof in an amount lessthan 5% w/w of the formulation.

80. A method according to claim 65, wherein the penetrant portion insaid formulation comprises a mixture of caprylic triglycerides andcapric triglycerides in amount less than 8% w/w of the formulation.

81. A method according to claim 65, wherein the penetrant portion insaid formulation comprises phosphatidyl choline in amount less than 12%w/w of the formulation.

82. A method according to claim 65, wherein the penetrant portion insaid formulation comprises a phospholipid in amount less than 12% w/w ofthe formulation.

83. A method according to claim 65, wherein the penetrant portion insaid formulation comprises a mixture of tridecane and undecane in amountless than 5% w/w of the formulation.

84. A method according to claim 65, wherein the penetrant portion insaid formulation comprises cetyl alcohol in amount less than 5% w/w ofthe formulation.

85. A method according to claim 65, wherein the penetrant portion insaid formulation comprises benzyl alcohol in an amount less than about 5w/w.

86. A method according to claim 65 wherein the penetrant portion in saidformulation comprises stearic acid in an amount less than 5% w/w of theformulation.

87. A method according to claim 65, wherein said formulation comprises agelling agent in an amount less than 5% w/w of the formulation.

88. A method according to claim 65 wherein the detergent portion in saidformulation comprises a nonionic surfactant in an amount between about2-25% w/w of the formulation; and a polar solvent in an amount less than5% w/w of the formulation.

89. A method according to claim 81, wherein the nonionic surfactant insaid formulation is a poloxamer and the polar solvent is water, analcohol, or a combination thereof.

90. A method according to claim 81, wherein the detergent portion insaid formulation comprises poloxamer, propylene glycol, glycerin,ethanol, 50% w/v sodium hydroxide solution, or a combination thereof.

91. A method according to claim 65, wherein the detergent portion insaid formulation comprises glycerin in an amount less than 3% w/w of theformulation.

92. A method according to claim 65, wherein the carbonate salt in saidformulation is sodium carbonate and/or sodium bicarbonate milled to aparticle size is less than 200 μm.

93. A method according to claim 65, wherein the carbonate salt is sodiumcarbonate and/or sodium bicarbonate in said formulation is milled to aparticle size is less than 70 μm.

94. A method according to claim 65, wherein the carbonate salt in saidformulation is sodium carbonate and/or sodium bicarbonate milled to aparticle size is less than 70 μm, wherein the sodium bicarbonate issolubilized in the formulation in an amount less than 20% w/w of theformulation.

95. A method according to claim 65, wherein the carbonate salt in saidformulation is sodium carbonate and/or sodium bicarbonate milled to aparticle size is less than 70 μm, wherein particle sizes less than about10 μm have an enhanced penetration thru the skin of a subject.

96. A method according to claim 65, wherein said formulation furthercomprises tranexamic acid in an amount less than 5% w/w of theformulation.

97. A method according to claim 65, wherein said formulation furthercomprises a polar solvent in an amount less than 5% w/w of theformulation.

98. A method according to claim 65, wherein said formulation furthercomprises a humectant, an emulsifier, an emollient, or a combinationthereof 99. A method according to claim 65, wherein said formulationfurther comprises ethylene glycol tetraacetic acid in an amount lessthan about 5 w/w.

100. A method according to claim 65, wherein said formulation furthercomprises almond oil in an amount less than about 5% w/w.

101. A method according to claim 65, wherein said formulation furthercomprises a mixture of thermoplastic polyurethane and polycarbonate inan amount less than about 5 w/w.

102. A method according to claim 65, wherein said formulation furthercomprises phosphatidylethanolamine in an amount less than about 5% w/w.

103. A method according to claim 65, wherein said formulation furthercomprises an inositol phosphatide in an amount less than about 5 w/w.

104. A method of evaluating a formulation for the treatment for canceror a proliferative disorder related to cancer, the method comprisingadministering a formulation for transdermal delivery through the skin ofa subject comprising one or more buffering agent, wherein saidadministration is evaluated for effectiveness to i) inhibit or preventthe metastasis of tumors or cancer cells, ii) inhibit or prevent thegrowth of a tumor or tumor cells, iii) inhibit or preventcarcinogenesis, iv) inhibit or prevent the intravasation of tumor cells,or v) improve or extend the duration of remission, or maintain remissionof a cancer or tumor.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe invention are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

1. A method of treating a melasma, comprising administering atransdermal formulation comprising a buffering agent to a patientsuffering from a melasma, wherein the transdermal formulation reducesthe level of brown to gray-brown at the site of melasma by at least 50%,wherein the buffering agent consists of sodium bicarbonate.
 2. Themethod of claim 1, wherein the transdermal formulation is administeredto a patient topically.
 3. The method of claim 1, wherein, thetransdermal formulation is effective in reducing symptoms of the patientsuffering from melasma.
 4. The method of claim 1, wherein thetransdermal formulation reduces an amount of skin pigmentation at thesite of the melasma.
 5. The method of claim 1, wherein the transdermalformulation reduces the level of brown to gray-brown patches at the siteof melasma by at least 60%.
 6. The method of claim 1, wherein thetransdermal formulation reduces the level of brown to gray-brown patchesat the site of the melasma by at least 65%.
 7. The method of claim 1,wherein the sodium bicarbonate is at a concentration from about 30% to35% w/w of the transdermal formulation.
 8. The method of claim 1,wherein the transdermal formulation comprises the buffering agent in anamount between about 10%-45% w/w; a penetrant portion in an amountbetween about 5%-55% w/w; a detergent portion in an amount between about1% to 15% w/w; and water in an amount between about 15% to 65% w/w. 9.The method of claim 1, wherein the transdermal formulation also includesone or more other therapeutic agents.
 10. The method of claim 1, whereinthe buffering agent penetrates the skin of the patient.
 11. The methodof claim 1, wherein the sodium bicarbonate is at a concentration fromabout 1.0% to 3.0% w/w of the transdermal formulation.
 12. The method ofclaim 11, wherein the transdermal formulation further comprises atranexamic acid.
 13. The method of claim 12, wherein the tranexamic acidis at a concentration from about 6.0% to 8.0% w/w of the transdermalformulation.
 14. The method of claim 13, wherein the transdermalformulation is formulated as a cream, lotion or ointment.
 15. A methodof treating a melasma, comprising administering a transdermalformulation comprising a buffering agent to a patient suffering from amelasma, wherein the transdermal formulation reduces the level of brownto gray-brown at the site of melasma by at least 50%, wherein thebuffering agent consists of sodium carbonate.
 16. The method of claim15, wherein the sodium carbonate is at a concentration from about 1.0%to 3.0% w/w of the transdermal formulation.
 17. The method of claim 16,wherein the transdermal formulation further comprises a tranexamic acid.18. The method of claim 17, wherein the tranexamic acid is at aconcentration from about 6.0% to 8.0% w/w of the transdermalformulation.
 19. The method of claim 18, wherein the transdermalformulation is formulated as a cream, lotion or ointment.